Graft-versus-host disease (GVHD) is a common complication of allogeneic hematopoietic cell transplantation (HCT) and is associated with significant morbidity and mortality. For many years, there have been few effective treatment options for patients with GVHD. First-line systemic treatment remains corticosteroids, but up to 50% of patients will develop steroid-refractory GVHD and the prognosis for these patients is poor. Elucidation of the pathophysiological mechanisms of acute and chronic GVHD has laid a foundation for novel therapeutic approaches. Since 2017, there have now been 4 approvals by the US Food and Drug Administration (FDA) for GVHD. Ruxolitinib, an oral selective JAK1/2 inhibitor, received FDA approval for the treatment of steroid-refractory acute GVHD in 2019 and remains the only agent approved for acute GVHD. There are currently 3 FDA approvals for the treatment of chronic GVHD: (1) ibrutinib, a BTK inhibitor traditionally used for B-cell malignancies, was the first agent approved for chronic GVHD after failure of one or more lines of systemic therapy, (2) belumosudil, an oral selective inhibitor of ROCK2, for patients with chronic GVHD who received at least 2 prior lines of treatment, and (3) ruxolitinib for chronic GVHD after failure of one or two lines of systemic therapy. In this review, we highlight the clinical data which support these FDA approvals in acute and chronic GVHD with a focus on mechanism of actions, clinical efficacy, and toxicities associated with these agents. Considering the increasing number of allogeneic hematopoietic stem cell transplantations now being performed each year, non-transplant oncologists are more likely to encounter patients receiving treatment for graft versus host disease (GVHD) in their clinical practice. This review highlights recent approvals of ruxolitinib, ibrutinib, and belumosudil as therapeutic agents for GVHD, with a focus on mechanism of action, efficacy, and toxicity of these agents. Graft-versus-host disease (GVHD) is a common and morbid complication of allogeneic hematopoietic cell transplantation (HCT). After many years without effective therapies, there have been 4 US Food and Drug Administration (FDA) approvals for GVHD treatment since 2017. In this review, we detail the efficacy and toxicity data from the clinical trials which support these approvals. As these newly approved agents are likely to be increasingly used in the clinical setting, this review has significant relevance for practicing oncologists treating patients after allogeneic HCT.1-3 Additionally, many patients with GVHD suffer impairments in psychosocial function that result in significantly reduced quality of life (QoL).5 GVHD occurs when alloreactive donor cells (the graft) recognize the transplant recipient’s body (the host) as foreign, resulting in immunologically mediated damage to host tissues.6Allogeneic hematopoietic stem cell transplantation (HCT) is a potentially curative treatment for numerous malignant and nonmalignant hematologic and immunological diseases. However, HCT has traditionally been reserved for high-risk disease due to the risks associated with treatment. Graft-versus-host disease (GVHD), which affects 30%-50% of patients, is a major cause of morbidity and mortality in allogeneic HCT recipients.7 Following preclinical investigations which further elucidated the pathophysiological mechanisms of both acute and chronic GVHD, the therapeutic landscape has shifted toward to the use of targeted agents with the hope of achieving improved clinical responses with less off-target effects.8-10 Since 2017, there have been 4 approvals by the US Food and Drug Administration (FDA) for GVHD: ruxolitinib (acute and chronic GVHD), ibrutinib (chronic GVHD), and belumosudil (chronic GVHD)  . Given tAcute and chronic GVHD have unique pathophysiologies, clinical presentations, and risk factors, which are highlighted in 13 While some aspects of the acute GVHD immune response overlap with chronic GVHD, the pathophysiology of chronic GVHD is distinct and can be summarized as having 3 phases: (1) host tissue injury which leads to early inflammation, (2) chronic inflammation and dysregulation of T-cell and B-cell immunity, and (3) tissue repair with fibrosis.11 Chronic GVHD biology is characterized by B-cell signaling and prolonged immune activation of T-cell subsets, regulatory T-cell deficiencies, and tissue fibrosis.8 Differences in the pathophysiological mechanism of acute and chronic GVHD form the basis for the development of unique prophylactic and therapeutic approaches to the disease.Generally, the pathophysiology of acute GVHD is thought to be initial injury to the recipient tissue, either from the conditioning regimen or infection, which ultimately leads to activation of alloreactive donor T cells which mediate tissue damage via direct attack or through propagation of inflammation. Acute GVHD is driven by recognition of mismatched major and minor histocompatibility antigens, altered mechanisms of tissue repair and protection, and loss of protective microbial-derived metabolites.14 The cardinal organ manifestations of acute GVHD are the skin (maculopapular rash), liver (hyperbilirubinemia and jaundice), and the gut . Although chronic GVHD may involve any organ system, the most common organs involved are the skin, eyes, and mouth. One feature unique to chronic GVHD is the development of fibrotic changes, most notably sclerodermatous GVHD and bronchiolitis obliterans syndrome.15While acute GVHD classically presents earlier after HCT than chronic GVHD, the distinction is defined by the symptoms at clinical presentation, rather than time of onset.12 Grading of disease severity remains clinically based with clear consensus criteria published for use in practice as well as in clinical trials for eligibility and response assessment.17 While noninvasive biomarkers are commercially available for acute GVHD risk stratification,19 no clinical trials have proven that these biomarkers should dictate choice of therapy. Chronic GVHD biomarkers are being actively investigated; however, none have yet emerged as being able to accurately risk stratify patients.20Proven clinical risk factors for acute GVHD include degree of recipient human leukocyte antigen (HLA) mismatching, use of total body irradiation, and increased intensity of the conditioning regimen while risk factors more associated with chronic GVHD include older age, female donors to male recipients, prior history of acute GVHD, and peripheral blood stem cells as a graft source.21-23 However, approximately 50% of patients become steroid-resistant or refractory (SR),24 and these patients have a dismal long-term prognosis, with an estimated 40% nonrelapse mortality (NRM) rate within 12 months.18 Till date, no consensus has been reached regarding the optimal approach for the management of SR acute GVHD; however, the FDA approval of ruxolitinib for SR acute GVHD in May 2019 provides an avenue to a widely adopted approach to the initial management of SR acute GVHD.25The standard approach to initial systemic treatment for acute GVHD is corticosteroids usually at a dose of 1-2 mg/kg/day of prednisone or its equivalent.26 The JAK signaling pathway is an important contributor of cytokine-driven tissue damage in acute GVHD, and preclinical models showed a reduction in incidence and severity of GVHD with the administration of JAK 1/2 inhibitors.27-30 The mechanism of immunomodulation induced by ruxolitinib is hypothesized to be via decreased neutrophil migration in the first phase of acute GVHD, decreased T-cell priming via downregulation of MHC-II and reduced cytokine release in the second phase, and reduced T-cell expansion in the third phase.31 The SR acute GVHD approval for ruxolitinib was based the results of REACH1, an open-label single-arm phase II trial which accrued subjects from December 2016 until July 2018 , which was defined as a complete response (CR), very good partial response (VGPR), or partial response (PR).REACH1 was a multicenter phase II trial which accrued subjects at 26 medical centers in the US. Eligible subjects included those who were at least 12 years of age with grades II-IV SR-GVHD per Mount Sinai Acute GVHD International Consortium (MAGIC) criteria,n = 48, 67.6%) had grade III-IV acute GVHD at enrollment. The median dose of corticosteroids at enrollment was 156.3 mg/day (range: 50-300) and the median duration of corticosteroid treatment prior to enrollment was 15 days (range: 3-285). The median average total daily dose of ruxolitinib was 10.3 mg/day (range: 5-20) and the median duration of treatment was 46 days (range: 4-473). The most common reasons for treatment discontinuation were adverse events , investigator discretion (28.2%), and death (9.9%). Only 8.5% of participants discontinued treatment for acute GVHD progression. More than half of subjects (54.9%) had a response at day 28, including 26.8% who experienced a CR. The median duration of response (DOR) was 345 days after ruxolitinib initiation. The ORR at any time for the entire cohort was 73.2%, including 4 subjects who responded after day 28 . The only significant association with response in subgroup analysis was GVHD grade at enrollment, as participants with grade II GVHD had higher ORR compared to grade III and IV (82.6% vs. 41.2% vs. 42.9%). Indeed, there was a correlation in CR rate from participants with baseline grade II GVHD (47.8%) to grade III (20.6%) and grade IV (7.1%). Responses were observed across type of organs involved in GVHD; however, subjects with 2+ organs involved were less likely to have a response at 28 days compared to participants with 1 organ involved at enrollment (47.2% vs. 62.9%). The median overall survival (OS) was 7.6 months, with 6- and 12-month OS rates of 51% and 42.6%, respectively. The 6-month cumulative incidence rate for nonrelapse mortality (NRM) was 44.4%  and the 12-month cumulative rate for NRM was 52.9%  and both were lower for day 28 responders. Only 4 subjects were reported to have developed chronic GVHD after treatment with ruxolitinib.Approximately 71 subjects received at least 1 dose of ruxolitinib. The median age was 58 years and most participants (n = 1).Every participant enrolled onto the study experienced at least 1 treatment-emergent AE and 74.6% experienced at least 1 treatment-related AE with the most common being thrombocytopenia , anemia , and decreased neutrophil count . Other AEs experienced by at least 10% of subjects included decreased white blood count  and alanine aminotransferase (ALT) elevation . Toxicity led to ruxolitinib discontinuation, dose reduction, and treatment interruption in 32.4%, 35.2%, and 40.8% of participants, respectively. Grade 5 AEs were experienced by 2 patients .P < .001) and were more likely to experience a CR (34% vs. 19%). Participants with grade II GVHD were most likely to experience an objective response in both groups . Subjects with grade IV GVHD receiving ruxolitinib were more than twice as likely to have a response compared to the control group . The DOR at 56 days was also significantly higher in the ruxolitinib group compared to the control group  and the best overall response at day 28 was 82% in the ruxolitinib group and 61% in the control group . Median OS and failure-free survival (FFS) also favored ruxolitinib .In primary endpoint analysis, subjects who received ruxolitinib had significantly higher day 28 ORR compared to the control group : 2.64, Nearly all patients who received ruxolitinib (95%) experienced at least 1 AE, with 78% experiencing grade ≥3 AEs. Hematologic laboratory abnormalities were noted with similar frequency as in REACH1 with thrombocytopenia , anemia , and neutropenia  being the most common. Other AEs observed in ≥10% of participants included cytomegalovirus infection, peripheral edema, hypokalemia, hypertension, hypoalbuminemia, pyrexia, and hypomagnesemia. AEs led to dose modification in 38% of subjects treated with ruxolitinib and 11% of participants discontinued ruxolitinib for AEs.35The results from REACH1 and REACH2 demonstrated that ruxolitinib is an effective treatment for SR acute GVHD with a tolerable toxicity profile. More than half of the subjects enrolled on these studies experienced an objective response at day 28 after treatment initiation (54.9%-62%), including more than one-quarter of subjects who experienced a CR (26%-33%). While REACH1 led to the FDA approval of ruxolitinib, REACH2 demonstrated significant improvement in efficacy outcomes with the use of ruxolitinib as compared to other current treatment options. We believe these studies established ruxolitinib as the standard of care for SR acute GVHD. However, real-world experiences with ruxolitinib will be of high interest. In practice, ruxolitinib may be initiated early as a steroid-sparing agent, before SR criteria from the clinical trials are met. Additionally, patients with grade III-IV disease at the time of enrollment on REACH2 only showed a 53%-56% response rate with ruxolitinib, illustrating that severe disease remains a significant unmet need. Future studies are needed to identify which patients are most likely to benefit from ruxolitinib, duration of therapy, how to taper and discontinue therapy, how to define ruxolitinib refractory disease and optimal treatments for those patients who do not have a satisfactory response to ruxolitinib.36 Although there is no consensus treatment for SR chronic GVHD, commonly used agents include calcineurin inhibitors, extracorporeal photopheresis (ECP), mammalian target of rapamycin (mTOR) inhibitors, rituximab, and mycophenolate mofetil. There have been 3 recent approvals for the treatment of refractory chronic GVHD: ibrutinib, belumosudil, and ruxolitinib.Similar to acute GVHD, corticosteroids have traditionally been the recommended first-line treatment for chronic GVHD.37 Pre-clinical models found that mice who underwent HCT from BTK- or ITK-deficient donors did not develop chronic GVHD, suggesting a possible role for ibrutinib for the treatment of chronic GVHD.38 Ibrutinib became the first FDA-approved agent for chronic GVHD in 2017 based on the results of a single-arm Phase II trial (PYC-1129) , which inhibits signal transduction from the B-cell receptor, activation of B-cells, and interleukin-2-inducible T-cell kinases (ITK).YC-1129) .40This clinical trial enrolled subjects across 11 centers in the US. Participants were eligible for the trial if they were ≥18 years old with steroid-dependent or SR chronic GVHD and had ≤ 3 prior systemic treatment regimens. Steroid-dependent disease was defined as GVHD requiring at least 12 weeks of prednisone ≥ 0.25 mg/kg, while SR disease was defined as progression of chronic GVHD after at least 4 weeks of treatment with ≥ 0.5 mg/kg prednisone. Patients were eligible if they had active chronic GVHD, defined as having an erythematous rash involving at least 25% of body surface area or a NIH mouth score >4. The requirement of one of these inflammatory manifestations of chronic GVHD has not been adopted in subsequent trials. The primary efficacy endpoint for the phase II portion of the trial was best ORR at any time, defined as a CR or PR based on the 2005 NIH Chronic GVHD Consensus Panel and modified based on the 2014 NIH response criteria.41 The ORR for the entire cohort was 67%, including 21% of participants who experienced a CR. Of the subjects who responded, 79% showed a response at their first assessment and 71% experienced a sustained response for ≥ 20 weeks. Organ-specific subgroup analysis demonstrated high response rates in the skin (88%) and mouth (99%). Participants with steroid-dependent GVHD had higher response rates compared to SR or both steroid-dependent and SR subjects (75% vs. 50% vs. 50%). One-year follow-up data showed 55% of patients had sustained responses for >44 weeks.41 The median corticosteroid dose decreased on ibrutinib from a baseline of 0.29-0.12 mg/kg at week 49 and 5 participants discontinued steroids during treatment with ibrutinib. Nearly one-quarter of subjects (24%) experienced at least a 7-point decrease in the Lee Symptoms Scale (LSS).A total of 42 subjects received 420 mg ibrutinib daily. The median age was 56 years and most participants previously received nonmyeloablative transplants (57%). The median time from transplant to chronic GVHD diagnosis was 7.6 months and the median time from chronic GVHD diagnosis to treatment with ibrutinib was 13.7 months. Most subjects (57%) had 2 organs involved in GVHD and 28% had ≥3 organs involved. The median time on treatment with ibrutinib was 4.4 months.n = 24, 57%), diarrhea , and muscle spasms . The most common serious adverse events (grade ≥ 3) were fatigue , pneumonia , and diarrhea . The only cardiac toxicity was atrial fibrillation, which was reported in one patient. Infectious complications were seen in 69% of participants and 2 subjects experienced fatal treatment-related AEs . Toxicity led to dose reductions and treatment discontinuation in 31% and 33% of participants, respectively.The majority of treatment-related AEs were grade 1-2 and the most common were fatigue  of 2 different dosing schedules of belumosudil , which is an important signaling pathway that regulates Th17/regulatory T-cell balance and the profibrotic pathway.umosudil .45This clinical trial enrolled subjects at 28 centers across the US. Important eligibility criteria included age ≥ 12 years, ongoing chronic GVHD manifestations, 2-5 prior lines of systemic therapy, and stable dose of corticosteroids for 2 weeks prior to screening.In total, 132 subjects were enrolled onto the study and randomized 1:1 to receive either 200 mg QD or 200 mg BID of belumosudil. Subjects were treated until unacceptable toxicity or clinically significant progression of chronic GVHD on treatment. Best ORR at any time, defined as the rate of participants experiencing either CR or PR per 2014 NIH Consensus Criteria was the primary endpoint of the study.n = 7). The ORR was similar for participants who received prior ibrutinib  or ruxolitinib . During treatment with belumosudil, 65% of subjects reduced their corticosteroid dose. Additionally, a clinically meaningful improvement in the LSS summary score from baseline was observed in 59% and 62% of subjects in the 200 mg daily and 200 mg twice daily cohorts.The median age of enrolled subjects was 56 years and the majority of subjects had either moderate (31%) or severe (67%) GVHD at the time of enrollment. More than one-half (52%) of participants had ≥ 4 organs involved. In this heavily pre-treated population, 72% of subjects received ≥ 3 prior lines of systemic therapy including 34% who received ibrutinib and 29% who received ruxolitinib. The median duration of belumosudil treatment was 10 months, with 44% staying on treatment for ≥ 1 year. The most common reasons for treatment discontinuation were progression of disease (15.9%), AEs (12.1%), voluntary withdrawal (9.8%), and physician discretion (8.3%). The ORR was similar between the QD  and BID dosing groups . The CR rate of all subjects was 5.3% , diarrhea (33%), nausea (31%), and cough (28%). Liver-related AEs were also reported in 24% of participants. Grade ≥3 AEs included pneumonia (8%), hypertension (6%), and hyperglycemia (5%).46Ruxolitinib was also recently granted FDA approval for SR chronic GVHD based on results from a phase III randomized control trial of ruxolitinib versus investigator’s choice for SR chronic GVHD (REACH 3) .46Participants were enrolled onto this international trial at 49 centers across 28 countries in the US, Europe, Asia, Canada, and Australia. Eligible subjects were ≥ 12 years of age who had previously undergone allogenic HCT and subsequently developed moderate-to-severe SR or steroid-dependent chronic GVHD per NIH consensus criteria. Subjects were excluded if they received 2 or more systemic therapies for chronic GVHD in addition to steroids and they were eligible if they previously received a JAK inhibitor for acute GVHD if they met the following criteria: (1) had a PR or CR to prior JAK inhibitor treatments and (2) JAK inhibitor had been discontinued at least 8 weeks prior to enrollment onto the trial. The primary endpoint was objective response at 24 weeks, defined as a CR or PR per NIH criteria.P < .001). More subjects in the ruxolitinib group had a CR compared to the control therapy group (6.7% vs. 3.0%). The best overall response was also significantly higher in the ruxolitinib group compared to the control group . Participants receiving ruxolitinib also had longer FFS  and higher Modified LSS response rate  compared to the control group. The ruxolitinib group was more likely to discontinue treatment for toxicity (17.0% vs. 4.9%) and less likely to discontinue for lack of efficacy (14.5% vs. 42.7%).In total, 329 subjects were randomized. Most participants (61.1%) were male and the median age was 49 years. More than half (56.5%) had severe disease, while 42.9% had moderate disease. The most common investigator choice agents used as control therapy were ECP (34.8%), mycophenolate mofetil (22.2%), and ibrutinib (17.1%). The ORR at 24 weeks was significantly higher in the ruxolitinib group compared to best available therapy , thrombocytopenia (21.2%), and neutropenia (10.9%). Other AEs experienced by >10% of subjects were pneumonia (10.9%), diarrhea (10.3%), ALT elevation (15.2%), elevated creatinine (13.9%), hypertension (15.8%), pyrexia (15.8%), cough (10.3%), and fatigue (10.3%).47 Furthermore, questions remain about how these agents will be used in clinical practice. Clinical trials till date have not allowed combined administration of these novel agents, which will inevitably occur. Reports on the safety and clinical efficacy of this approach will be of particular interest.The FDA approvals of ibrutinib, belumosudil, and ruxolitinib over the last 5 years represent a monumental achievement for chronic GVHD therapeutics that will hopefully translate into improved clinical outcomes for patients. All 3 agents have shown clinically meaningful responses, characterized by high ORRs (67%-76%) and improvements in QoL. CRs have been generally rare in these studies, due to the nature of chronic GVHD itself. Ibrutinib had the highest reported CR rate (21%), although this may be in part due to more than half of the subjects in that trial having only 2 organs involved in their GVHD and cross-trial comparison is not appropriate due to differences in patient selection. Thus, we believe there is no consensus standard of care regarding choice of therapy for SR chronic GVHD, given the lack of head-to-head comparison . Practic48 BMT CTN 1705 is an ongoing randomized phase III trial that will compare the use of AAT with corticosteroids to corticosteroids alone as first-line therapy for subjects with high risk acute GVHD (NCT04167514). T-guard, a combined CD3/CD7 immunotoxin which can inhibit both activated T-cell and NK cell function, will be compared to ruxolitinib in a randomized phase III trial in the treatment of SR Grade III-IV acute GVHD .49 Similarly, a phase III trial of itolizumab, a monoclonal antibody targeting CD6-ALCAM pathway, in the up-front treatment of acute GVHD is planned, after demonstrating preliminary safety and efficacy in a phase Ib/II study.50 Axatilimab, a monoclonal antibody that targets CSF1-R to address aberrant macrophage function, is being investigated as therapy for SR chronic GVHD. Following a phase I/II study demonstrating tolerability and efficacy,51 an international, randomized phase II registration study for subjects with refractory chronic GVHD who have received at least 2 prior treatments has begun enrollment (NCT04710576). Aside from immune-targeted approaches to GVHD, the microbiome has emerged as a target for intervention, as lower intestinal microbial diversity during allogeneic HCT is associated with higher mortality and risk of death from acute GVHD.53 Fecal microbiome transplantation (FMT) can reverse dysbiosis after transplant,54 and preliminary studies have shown this approach to be clinically effective in treating SR acute GVHD.56 Although infection transmission is a possible risk with FMT,57 these events are likely rare.58 Larger multicenter studies are ongoing and planned to better evaluate the clinical efficacy of this microbiome-targeted approaches for SR acute GVHD .Ongoing studies for GVHD are investigating novel agents, combination therapies, and steroid-sparing approaches. Given the numerous pathophysiologic mechanisms at play in both acute and chronic GVHD, there is a great opportunity to develop novel treatments, which remain an unmet need for this population. While many clinical trials are ongoing, a few select agents are being investigated in larger studies. Alpha-1-Antitrypsin (AAT), a serine protease inhibitor with anti-inflammatory and immunomodulatory properties, has previously demonstrated good tolerability and clinical efficacy as a treatment for SR acute GVHD.59 Similarly, in chronic GVHD, the addition of ibrutinib in the front-line treatment of moderate/severe disease failed to improve response rates as compared to placebo when used in combination with corticosteroids (NCT02959944).60 An ongoing phase III trial is randomizing patients with newly diagnosed moderate or severe chronic GVHD to treatment with itacitinib or placebo in combination with corticosteroids (NCT03584516).While many clinical trials have targeted SR-GVHD, therapeutic agents are now being investigated in the upfront setting, acknowledging the suboptimal response to corticosteroids. Three recent large clinical trials have added investigational agents to corticosteroids in the front-line setting, in addition to BMT CTN 1705 mentioned above. In a randomized phase III trial, the addition of itacitinib, a selective JAK1 inhibitor, to corticosteroids did not significantly improve Day 28 ORR for grade II-IV acute GVHD as compared to corticosteroids with placebo (NCT03139604).58 In BMT CTN 1501, sirolimus demonstrated similar treatment efficacy and overall lower toxicity profile as compared to prednisone for standard risk GVHD, defined by the Minnesota GVHD Risk Score and MAGIC biomarker status.61 There are currently 2 clinical trials investigating this approach in chronic GVHD: ibrutinib as monotherapy (NCT04294641) and the combination of itacitinib and ECP (NCT04446182). Additionally, select GVHD therapeutics are targeted to specific clinical manifestations. For example, in acute gastrointestinal GVHD, interventions such as FMT56 and anti-α4-integrins, such as natalizumab and vedolizumab,63 have demonstrated organ-specific clinical efficacy. Similar approaches are of high interest for patients with fibrotic manifestations of chronic GVHD.64Furthermore, clinical trials in GVHD are striving to develop more individualized approaches to treatment. In acute GVHD, combinations of clinical risk scores and blood biomarkers are now being used to risk stratify patients with either higher- or lower-risk disease. In the setting of high-risk GVHD, the addition of other therapeutic agents to systemic corticosteroids for upfront treatment is being investigated (NCT02133924). In patients with lower-risk acute GVHD, trials are now investigating whether specific agents can be used that limit corticosteroid use or avoid them altogether. The theoretical benefits of such studies include limiting the toxicity of corticosteroids and creating an opportunity to better elucidate the biological effect of targeted monotherapy.With an increasing number of allogeneic HCT being performed each year, nontransplant oncologists are more likely to be involved in the management of patients with ongoing GVHD. While the recommended frontline systemic therapy for both acute and chronic GVHD remains corticosteroids, there has long been no consensus on the treatment of SR-GVHD. There are now 3 agents approved for SR-GVHD: ruxolitinib (acute and chronic), ibrutinib (chronic), and belumosudil (chronic). The results of REACH2 make a compelling case that ruxolitinib should be considered the consensus first choice in the treatment for acute SR-GVHD and its uptake into clinical practice is increasing. In chronic GVHD, all 3 agents have shown good efficacy and favorable toxicity profiles in clinical trials. Future studies will continue to investigate these agents and other novel therapies in multiple settings and combinations and are sure to further advance the therapeutic landscape for GVHD.


C1-C3) were tested for their anticancer efficacy against 59 cancer cell lines in a single dose assay. 1--4,6-bis((E)-4-hydroxy-3-methoxystyryl)pyrimidin-2(1H)-one (C2) demonstrated the most promising anticancer activity with mean percent growth inhibition (%GIs) of 68.22 in single dose assay at 10 µM. The compound exhibited >68 %GIs against 31 out of 59 cancer cell lines and was found to be highly active against all leukemia and breast cancer cell lines. The compound C2 showed a lethal effect on HT29 (colon cancer) with %GI of 130.44, while 99.44 %GI was observed against RPMI-8226 (Leukemia). The compound C2 displayed better anticancer activity against the panels of CNS, melanoma, ovarian, prostate, and breast cancer cell lines than curcumin and other anti-EGFR agents gefitinib and imatinib in single dose assay. The compound C2 also demonstrated potent anticancer activity in a 5-dose assay (0.001 to 100 µM) with GI50 values ranging from 1.31 to 4.68 µM; however, it was found to be non-selective with SR values ranging from 0.73 to 1.35. The GI50 values of compound C2 were found to be better than that of the curcumin against all nine panels of cancer cell lines. All of the curcumin analogues were subsequently investigated for molecular docking simulation against EGFR, one of the most attractive targets for antiproliferative action. In molecular docking studies, all the ligands were found to accommodate the active site of EGFR and the binding affinity of ligand C2 was found to be −5.086 kcal/mol. The ligand C2 exhibited three different types of interactions: H-bond (Thr790 and Thr854), π-cationic (Arg841), and aromatic H-bond (Asn842). The curcumin analogues reported in the current investigation may provide valuable therapeutic intervention for the prevention and treatment of cancer and accelerate anticancer drug discovery programs in the future.Natural products (NPs) continue to provide a structural template for the design of novel therapeutic agents and expedite the drug discovery process. The majority of FDA-approved pharmaceuticals used in medical practice can be traced back to natural sources, and NPs play a significant role in drug development. Curcumin, one of the most well-studied chemicals among the NPs, is currently the subject of intense investigation for its biological effects, including the prevention and treatment of cancer. Cancer has overtaken all other causes of death in the world today, with 19.3 million new cases and nearly 10 million deaths predicted in 2020. In the present investigation, we reported the synthesis of three semi-synthetic analogues of curcumin-bearing pyrimidinone moiety by the chemical modification of the diketone function of curcumin followed by their characterization by analytical techniques including infrared (IR), nuclear magnetic resonance (NMR), and mass spectral data. According to the National Cancer Institute (NCI US) methodology, the curcumin analogues ( Curcuma longa Linn.), has been utilized to prepare a variety of semi-synthetic analogues [2-), aryl side chain, diketone group, and carbon-carbon double bonds (-CH=CH-) with improved bioactivity [Currently, a major challenge facing the modern scientific community is the development of novel anticancer drugs with fewer side effects. Research on natural products (NPs) has been boosted recently because NPs are thought to be relatively safer than synthetic ones ,2. The unalogues ,20,21,22activity ,24. In tactivity . Our resactivity ,27,28. Nactivity ,18,19,20Cancer has overtaken all other causes of death in the world today, with 19.3 million new cases and nearly 10 million deaths predicted in 2020 . NowadayCurcumin was isolated by a conventional method as per the reported procedure and nearly 250 mg of curcumin was isolated with 80 g of the ground tumeric powder .C1-C3) bearing pyrimidinone moiety were synthesized by conventional heating method and the synthetic protocol is summarized in A1)  and substituted phenyl urea (B1-B3) (0.20 mmol) in glacial acetic acid (10 mL) was stirred in a sandbath at 80 °C for 8 h. The reaction mixture was then concentrated and poured into the crushed ice to obtain crude final products (C1-C3). The crude product was then re-crystallized with ethanol to obtain 1-substitutedphenyl-4,6-bis((E)-4-hydroxy-3-methoxystyryl)pyrimidin-2(1H)-one (C1-C3). The completion of the reaction was monitored throughout by preparatory thin layer chromatography (TLC Silica gel 60 F254) in the mobile phase n-hexane: ethylacetate (6: 4). The substituted phenyl urea ((B1-B3) was prepared as per the reported method [The curcumin analogues  was carried out against 60 NCI cancer cell lines derived from nine different panels  at a single dose (10 µM) and five dose assay as per the National Cancer Institute US [C1-C3 in single dose assay at 10 µM are given in the C1 displayed more than 50 to 67 percent growth inhibitions (% GIs) against three leukemia cancer cell lines  with %GIs of 63.58, 57.03, and 55.82 respectively, and one breast cancer cell line MCF7 (%GIs of 52.38). The compound C1 showed moderate anticancer activity with %GI of 20–50 against 16 cancer cell lines HCT116 (%GI = 49.42), MDA-MB-231 (%GI = 39.69), LOX IMVI (%GI = 38.43), RPMI-8226 (%GI = 35.53), UO-31 (%GI = 35.12), BT-549 (%GI = 33.10), SNB-75 (%GI = 30.28), PC-3 (%GI = 27.72), MDA-MB-435 (%GI = 25.07), U251 (%GI = 24.47), SF-268 (%GI = 23.95), MDA-MB-468 (%GI = 23.56), KM12 (%GI = 23.01), SF-539 (%GI = 21.92), EKVX (%GI = 21.89), and OVCAR-4 (%GI = 21.66), while showed less activity against rest of the 37 cancer cell lines. The compound C1 displayed the most promising anticancer activity against IGROV1 with %GI of 91.43. The compound C1 displayed significant anticancer activity against the colon cancer cell line HT29 with %GI of 55.65, while displayed 40–50 %GIs against four cancer cell lines viz. NCI-H522 (%GI = 48.31), MOLT-4 (%GI = 47.47), RPMI-8226 (%GI = 44.65), and CCRF-CEM (%GI = 44.43). The compound C3 displayed 20–40 %GIs against 12 cancer cell lines, HCT-116 (%GI = 38.73), MDA-MB-231 (%GI = 34.16), MCF7 (%GI = 32.66), SR (%GI = 32.20), UACC-257 (%GI = 32.02), U251 (%GI = 27.95), UO-31 (%GI = 27.54), K-562 (%GI = 25.32), UACC-62 (%GI = 24.86), SN-12C (%GI = 23.96), LOX IMVI (%GI = 21.97), and A529 (%GI = 21.87), while it displayed less activity against the rest of the 42 cancer cell lines. The compounds C1 and C2 displayed lethal effects on the colon cancer cell line HT29 with %GI of 105.09 and 130.44 respectively. The compound C2 displayed the most promising anticancer activity among the series. The compound C2 displayed 50–67 %GIs against 11 cancer cell lines IGROV1 (%GI = 66.54), SNB-19 (%GI = 65.86), PC-3 (%GI = 65.14), HS-578T (%GI = 63.69), ACHN (%GI = 62.22), UACC-62 (%GI = 61.83), UACC-257 (%GI = 58.9), HCC-2998 (%GI = 58.23), A549 (%GI = 55.85), EKVX (%GI = 52.61), SK-OV-3 (%GI = 52.29), SNB-75 (%GI = 51.58), and NCI-H226 (%GI = 51.15), and displayed 20–50 %GIs against HOP-62 (%GI = 49.42), NCI/ADR-RES (%GI = 44.79), OVCAR-4 (%GI = 43.74), OVCAR-5 (%GI = 42.42), M14 (%GI = 42.29), MALME-3M (%GI = 41.21), CAKI-1 (%GI = 36.82), COLO 205 (%GI = 29.19), NCI-H460 (%GI = 29.16), NCI-H322M (%GI = 27.35), SF-295 (%GI = 25.63), A498 (%GI = 21.46), and HOP-92 (%GI = 21.31), while displaying less activity with %GIs <20 against 2 cancer cell lines namely SK-MEL-2 (%GI = 17.08) and TK-10 (%GI = 13.98). The compound C2 displayed the most promising anticancer activity with %GIs more >68 against 30 cell lines namely RPMI-8226 (%GI = 99.44), LOX IMVI (%GI = 97.17), SF-539 (%GI = 96.65), SR (%GI = 95.88), MDA-MB-435 (%GI = 95.43), HCT-116 (%GI = 95.37), OVCAR-8 (%GI = 92.47), U251 (%GI = 91.46), K-562 (%GI = 90.79), MOLT-4 (%GI = 90.29), NCI-H522 (%GI = 90.11), HCT-15 (%GI = 89.72), CCRF-CEM (%GI = 89.66), SW-620 (%GI = 89.57), OVCAR-3 (%GI = 89.34), RXF 393 (%GI = 88.18), KM12 (%GI = 87.88), MCF7 (%GI = 86.25), BT-549 (%GI = 84.83), SK-MEL-28 (%GI = 82.41), SF-268 (%GI = 80.15), T-47D (%GI = 75.89), UO-31 (%GI = 74.86), SN 12C (%GI = 72.16), DU-145 (%GI = 71.26), NCI-H23 (%GI = 71.04), MDA-MB-231 (%GI = 71.00), MDA-MB-468 (%GI = 70.41), 786-O (%GI = 70.30), and HL-60(TB) (%GI = 46.47). The anticancer activity of curcumin analogues (C1-C3) is shown in C1-C3 were compared with curcumin and other EGFR inhibitors, Gefitinib and Imatinib, and their comparative anticancer activity at 10 µM is given in C2 displayed better anticancer than curcumin and standard drug imatinib and gefitinib activity against CNS, melanoma, ovarian, prostate, and breast cancer cell lines. The curcumin analogues displayed mean growth percent inhibition of 68.22 (%GI = >68), and were further selected for screening in a 5-dose assay [The anticancer activity of the curcumin analogues  (GI50 = 2.64 µM) among leukemia cell lines, NCI-H522 (GI50 = 1.55 µM) among non-small cell lung cancer cell lines, HCT-116 (GI50 = 1.31 µM) among colon cancer cell lines, SF-539 (GI50 = 2.18 µM) among CNS cancer cell lines, MDA-MB-435 (GI50 = 1.63 µM) among melanoma cell lines, OVCAR-5 (GI50 = 2.76 µM) among ovarian cancer cell lines, CAKI-1 (GI50 = 1.46 µM) among renal cell lines, DU-145 (GI50 = 3.68 µM) among prostate cancer cell lines, and MCF7 (GI50 = 2.00 µM) among breast cancer cell lines. The compound C2 exhibited non-selectivity against all the nine panels of cancer cell lines with a selectivity ratio (SR) ranging between 0.73 and 1.35 as the value of SR was found to be less than three  including H-bond, π-cationic, and halogen bonds with a binding affinity of −4.936 to −5.117 kcal/mol. The ligand C1 showed an aromatic H-bond with the residue Asp855 ; halogen bond (with the residue Lys875); aromatic H-bond (with the residue Asp855)  . The ligC1-C3) were reported in the current work. Curcumin and substituted phenyl urea in glacial acetic acid were stirred at 80 °C for 8 h to obtain 1-aryl-4,6-bis((E)-4-hydroxy-3-methoxystyryl)pyrimidin-2(1H)-ones (C1-C3) in good yields (75–80%). All the title compounds were characterized by analytical techniques, followed by their anticancer evaluation and molecular docking studies. One of the curcumin analogues (C2) displayed the most promising anticancer activity with a mean GP of 31.78 (% GI = 68.22). Nearly 31 cancer cell lines were found to be highly sensitive against the compound C2 with %GIs of >68. Two of the curcumin analogues (C1 and C2) displayed lethal effects on HT29 colon cancer cell lines with %GIs of 105.09 and 130.44. The anticancer activity of compound C2 was found to be better than that of curcumin and anti-EGFR drugs (gefitinib and imatinib) against CNS, melanoma, ovarian, prostate, and breast cancer cell lines, it was also found as active against all the cell lines of leukemia and breast cancer cell line panels  was found to be promising more than the previously reported work [C3) substitution in the phenyl ring at 1-pyrimidine showed mean %GI of 14.33, while 4-chloro (C1) substitution showed mean %GI of 20.37 and 2,6-dichloro (C2) substitution showed the most promising anticancer activity with %GI of 68.22. Three types of interaction were observed for the ligands (C1-C3) including H-bond, π-cationic, and halogen bonds with a binding affinity of -4.936 to -5.117 kcal/mol. The ligands C1 and C2 bind to the EGFR active site efficiently, whereas the ligand C3 binds less efficiently. The most active compound (C2) showed H-bond interaction with residue Thr854 through the water molecule, π-cationic interaction with residue Arg841, and halogen bond interaction with the water molecule. It also showed good interaction with residues Ala743, Val726, Thr790, Lys745, Leu799, Asp800, Gly721, Leu781, Cys775, Leu844, Cys797, Ser720, and Phe723.Three new curcumin analogues   and substituted phenyl ureas (B1-B3) (0.20 mmol) in glacial acetic acid (10 mL) was stirred in a sand bath at 80 °C for 8 h. After completion, the reaction mixture was concentrated under a vacuum to remove excess solvent and poured into crushed ice, filtered, dried, and recrystallized with ethanol to yield compound C1-C3 ,26.An equimolar mixture of curcumin (C1): Mp 132–134 °C; IR (KBr) vmax: 3382 (OH), 1637 (C=O), 1558 (C=N), 1163 (O-CH3), 713 (C-Cl) cm−1; 1H NMR : δ 1H NMR : δ; 3.81 , 6.04 , 6.71–6.75 , 6.79–6.81 , 7.12–7.13 , 7.29 , 7.41 , 7.43 , 9.62 ; ESI-MS: 504.2 (M+1)+ 506.1 (M+2)+.(C2): Mp 148–150 °C; IR (KBr) vmax: 3402 (OH), 1638 (C=O), 1559 (C=N), 1153 (O-CH3), 713 (C-Cl) cm−1; 1H NMR : δ; 3.81 , 6.04 , 6.71–6.75 , 6.79–6.81 , 7.12–7.16 , 7.30 , 7.50–7.54 , 9.62 ; ESI-MS: 538.2 (M+1)+ 540.1 (M+2)+.(C3): Mp 164–162 °C; IR (KBr) vmax: 3382 (OH), 1638 (C=O), 1558 (C=N), 1154 (O-CH3), 875 (C-Br), 713 (C-Cl) cm−1; 1H NMR : δ; 3.81 , 6.04 , 6.69–6.71 , 6.77–6.79 , 6.81–6.99 , 7.11 , 7.22 , 7.42–7.44 , 9.62 ; ESI-MS: 522.1 (M+1)+ 524.1 (M+2)+.(C1-C3) was evaluated against nine diverse panels of 59 cancer cell lines at a single dose (10 µM) and a 5-dose assay (0.001 to 100 µM) according to the National Cancer Institute (NCI US) protocol [The antiproliferative activity of the curcumin analogues  X-ray crystal structure with a resolution of 3.00 Å; R-value 0.194 (observed) was obtained from the protein data bank (https://www.rcsb.org/structure/2j5f) [C1-C3 were saved as mol file and the docking was done as per the protocol reported [The molecular docking against EGFR was performed for the ligands, re/2j5f) . The ligreported .C1-C3) were synthesized in good yields and characterized by analytical data of IR, NMR, and mass spectroscopy. The anticancer activity of curcumin analogues was evaluated in a single-dose experiment at 10 µM. The anticancer activity of compound C2 was found to be promising with %GI of 68.22 percent, and superior to curcumin, gefitinib, and imatinib against CNS, melanoma, ovarian, prostate, and breast cancer cell lines. The compound C2 displayed promising anticancer activity and has been further evaluated in 5-dose assay displayed anticancer activity with GI50 values ranging from 1.31 to 4.68 µM; however, found to be non-selective with SR values ranging from 0.73 to 1.35. The compound C2 was found to be more active than curcumin against all nine panels of cancer cell lines. Since EGFR was found to be over-expressed in a number of cancer cell lines, including those from breast, colon, non-small cell lung, renal, melanoma, ovarian, and prostate cancers we selected EGFR as a potential target and the mechanism behind the anticancer activity of the title compounds. We further studied the binding insight of our compounds (C1-C3) against the active binding site EGFR where 6-acrylamido-4-anilinoquinazoline usually bind [C2 was found to be −5.086 kcal/mol. The ligand C2 exhibited three different types of interactions: H-bond (Thr790 and Thr854), π-cationic (Arg841), and aromatic H-bond (Asn842). Furthermore, the ligand C2 displayed good interaction with residues Ala743, Val726, Thr790, Lys745, Leu799, Asp800, Gly721, Leu781, Cys775, Leu844, Cys797, Ser720, and Phe723. The curcumin analogues reported in the current investigation may provide valuable therapeutic intervention for the prevention and treatment of cancer and accelerate anticancer drug discovery programs in the future.Curcumin was successfully isolated from the ground turmeric powder and chemically modified to prepare three semi-synthetic analogues bearing pyrimidinone nucleus. All the compounds (lly bind . In mole


Despite a range of emerging immunotherapeutic strategies, the aggressive nature of DLBCL poses an ongoing clinical challenge. Therefore, there is a need to pursue more effective treatment modalities based on monoclonal antibodies, antibody-drug conjugates and CAR-T cell therapy. Up to now, the above-mentioned immunotherapeutic options have been extensively studied with the aid of established cell line models, which have significantly facilitated proceeding from preclinical to clinical investigations and led to improvement of DLBCL treatment. However, there are still several important challenges associated with faithful recapitulation of the aggressive nature of DLBCL. Therefore, the current review discusses means in which cell line models fulfill an essential tool leading to greater understanding DLBCL biology and development of novel immunotherapeutic strategies.Despite the high incidence of diffuse large B-cell lymphoma (DLBCL), its management constitutes an ongoing challenge. The most common DLBCL variants include activated B-cell (ABC) and germinal center B-cell-like (GCB) subtypes including DLBCL with MYC and BCL2/BCL6 rearrangements which vary among each other with sensitivity to standard rituximab (RTX)-based chemoimmunotherapy regimens and lead to distinct clinical outcomes. However, as first line therapies lead to resistance/relapse (r/r) in about half of treated patients, there is an unmet clinical need to identify novel therapeutic strategies tailored for these patients. In particular, immunotherapy constitutes an attractive option largely explored in preclinical and clinical studies. Patient-derived cell lines that model primary tumor are indispensable tools that facilitate preclinical research. The current review provides an overview of available DLBCL cell line models and their utility in designing novel immunotherapeutic strategies. The most common non-Hodgkin lymphoma (NHL) subtype, diffuse large B-cell lymphoma (DLBCL), accounts for up to 40% of all lymphoid tumors . Its manIn 2000, Alizadeh et al. identified two main subtypes: germinal center B-cell (GCB)-like and activated B-cell (ABC)-like subtype, with the latter being characterized with significantly worse prognosis . DiscoveA subgroup of DLBCL with MYC, BCL2 and BCL6 rearrangement can be identified which is linked to worse prognosis ,8. DevelSince establishment of the first leukemia-lymphoma (LL) cell lines in 1964  a vast nImmortalized LL cell lines offer a repeatable and reliable diagnostic tool to model primary tumor in the clinical arena around the world . HoweverNoteworthy, cell line models often arise from patients with end-stage, non-nodal, and leukemic phase lymphomas that possess a wide-ranging repertoire of biased mutational sequences . TherefoIt is also common to perform experiments using cell line models representative of other B-cell malignancies and extrapolating them to DLBCL as certain features overlap between them ,27. The DLBCL biology is studied with highly heterogeneous models representative of specific subtypes . Cell liABC-DLBCL, which constitutes around 30% of DLBCL cases, is characterized with a clinically unfavorable outcome as compared to GCB-DLBCL . SeveralUnique features of the available cell lines allow for the choice of tailored preclinical models. For instance, RC-K8 with dysregulated Rel/NF-κB pathway is an appealing candidate for testing novel immunotherapeutic agents for ABC subtype . MoreoveGCB-DLBCL constitutes up to 50% of DLBCL cases and has a GC phenotype defined as CD10+, BCL6+ . AlthougInterestingly, Karpas422, bearing both t and t along with several other abnormalities, is an attractive model to study chemotherapy-resistant NHL as it originated from DLBCL patient unresponsive to consecutive chemotherapeutic schemes .One of the hallmarks of GCB-DLBCL, present in up to 30% of cases, is t translocation, which results in Bcl-2 overexpression ,40. AddiUnique cell line representatives of RS-DLBCL formed on CLL background and secondary DLBCL formed on Hodgkin lymphoma (HL) background have also been developed. RS-DLBCL represents a group of mainly CD5+ and CD23+ large blast-like neoplastic B cells with small nucleoli with diffuse growth pattern and high proliferative potential . Up to 9Generally, aggressive NHL has a worse prognosis in comparison to HL ,47. HoweDespite being an indispensable tool within cancer research, most of the cell lines fail to reliably recapitulate the importance of genetic and microenvironmental factors of tumors in disease progression . Thus, 3Duś-Szachniewicz et al. established 3D spheroid models based on Ri-1 and Raji cells, which were used to test cytotoxicity of doxorubicin (DOX) and ibrutinib (IBR) on B-NHLs . Co-cultYears of RTX employment as well as recent findings on mechanisms of insensitivity to a range of immunotherapies indicate that developing resistance is an unavoidable side effect of the regimens . GeneratRTX-resistant cell lines (RRCLs) were generated by Czuczman et al. from Raji, SU-DHL-4, RL and U-2932 . The celThe mechanisms of potential insensitivity to CD37-targeting agents can also be investigated in cell line models. Accordingly, Arribas et al. generated two DLBCL cell line models (SU-DHL-2 and SU-DHL-4), which were resistant to IMGN529/DEBIO1562 (an anti-CD37 ADC) and demonstrated different phenotypic changes within the models . SU-DHL-The availability of various lymphoma cellular models is vital for testing novel antibodies in preclinical trials. Several B-cell antigens e.g., CD19, CD22, CD37, CD40, CD47, CD79b, and CD80 have been proposed as targets for mAbs mainly for r/r patients .Gehlert et al. used B-ALL cell lines  to test efficacy of CD19-targeting mAb, tafasitamab . The mAbAs demonstrated in a range of preclinical studies with cellular models, the CD37 antigen is widely expressed across multiple types of B-cell lymphoid neoplasms , which pSince it has been shown that CD38 is an important prognostic marker also in DLBCL , preclinFurthermore, Bouwstra et al. demonstrated that high expression of CD47, the so-called “don’t eat me” immune checkpoint, correlated with detrimental effect on OS in non-GCB DLBCL patients after R-CHOP therapy . FurtherBy now, it has been discovered that PD-L1 is aberrantly expressed on HLs and little is known about its role in NHLs . AnalysiAntibody-drug conjugates (ADCs) are monoclonal antibodies bound to cytotoxic payload that is delivered directly to the tumor cells . ImmunolApproval of polatuzumab vedotin (Pola) in 2019, CD79b-targeting ADC, benefited a significant percentage of r/r DLBCL patients . NonetheMoreover, sensitivity of 27 commonly used DLBCL cell lines of ABC and GCB subtypes to MMAE-conjugated ADCs: anti-CD22 (pinatuzumab vedotin) and anti-CD79B ADCs (Pola) was assessed . AlthougFurthermore, a potent in vitro activity against multiple B cell lines  via ADCC, ADCP, and CDC was exerted by naratuximab emtansine (IMGN529), an investigational CD37-targeting ADC conjugated to DM1 . The resIntroduction of T cells modified with chimeric antigen receptors (CARs) has led to a significant advancement in management of multiple malignancies, including lymphoid neoplasms . By now,The great success of anti-CD19 CAR-T cells in B-ALL encouraged preclinical studies on their utility against DLBCL. Interestingly, improved killing of B-cell lymphoma lines, including OCI-Ly2 and OCI-Ly19, Raji, Daudi, DEL (anaplastic large cell lymphoma model), Granta-519 and Jeko-1 (MCL), was triggered by the combination of anti-CD19-CAR-T cells with an anti-CD20-IFN fusion protein indicating that antibody-targeted IFN could improve the CAR-T cell therapy . NoteworFurthermore, promising findings on anti-CD37 mAbs also encouraged exploration of CD37 as a target for CAR-T cells. The panel of several B-cell lymphoma models  and two xenograft mice models were constructed to analyze the cytotoxic effect of CD37CAR .Despite the promising results on CD19 CAR-T cells, around half of high-grade lymphoma patients develops resistance after receiving such treatment . UnfortuBoth Animal models are a powerful tool to test novel immunotherapeutic options prior to clinical investigations . In esseMurine models are commonly utilized in testing efficacy and safety of mAbs, ADCs and CAR-T cells. Xenograft models derived from different cell lines injected subcutaneously  demonstrated significant lymphoma inhibition by XmAb5574, an anti-CD19 mAb with an Fc-engineered domain for effector function improvement. . FurtherThe current review has summarized available cell lines used as powerful tools in investigating DLBCL biology as well as in evaluating therapeutic efficacy of antitumor agents. Despite the wide arsenal of cellular DLBCL models representative of ABC and GCB subtypes, only several are repeatedly used, including SU-DHL-4, SU-DHL-6, OCI-Ly-3, TDM8, or U-2932, to optimize available immunotherapies. To ensure reliable modeling of the primary tumor it is crucial to analyze a genetic background of each model and ensure it remains uncontaminated with foreign material. Importantly, several lines have been authenticated and included in the LL-100 panel .Effective employment of cell line models encourages other research teams to utilize the same model systems, which on the one hand facilitates the reproducibility of the generated data among research centers yet limits the scope of investigations. After all, DLBCL remains one of the most heterogenic lymphoid malignancies and requires multiple cellular models to study its biology. Simultaneously, it is crucial to report failures associated with culturing of specific cell lines to allow for verification and perhaps modifications of the models to overcome culturing challenges. Surprisingly, U-2932 remains a frequently used cell line in DLBCL research regardless of the existing controversies on its complex phenotype, which might perhaps serve as an advantage in development of more universal immunotherapeutics. Furthermore, it remains a common strategy to use other B-cell lymphoma models, especially BT cell lines  to yield results and extrapolate them to DLBCL.Importantly, the wide arsenal of the existing cellular models contributed significantly to the acceleration of the preclinical testing of various immunotherapeutics, which has been observed in the past decade along with approval of a range of mAbs, ADCs, and CAR T. However, years of immunotherapy employment demonstrated that it inevitably leads to development of resistance, which remains an ongoing challenge to overcome. Consequently, an urge to expand even wider arsenal of commonly available patient-derived cell lines and to develop agent-resistant cell lines persists. Furthermore, investment in 3D systems is required to provide insight into efficacy of anti-tumor molecules before proceeding with animal testing. The attempts aiming at optimizing modeling of the primary tumor will benefit in improved optimization of (immuno)therapeutic strategies.


Nanoparticle treatment on tumor cells is proposed for its potential radiosensitizing properties, increasing the radiation effect on tumor cells and reducing the adverse effects on healthy tissues. The present study evaluates, on two cell lines derived from colon and breast adenocarcinomas, the impact of irradiation in the presence of specifically targeted gold nanoparticles. Cells were irradiated in the absence and in the presence of non-functionalized or specifically functionalized gold nanoparticles. The results pointed out that actively targeting gold nanoparticles has a clear radiosensitizing effect in both cell lines.Radiation therapy is widely used as an anti-neoplastic treatment despite the adverse effects it can cause in non-tumoral tissues. Radiosensitizing agents, which can increase the effect of radiation in tumor cells, such as gold nanoparticles (GNPs), have been described. To evaluate the radiosensitizing effect of 50 nm GNPs, we carried out a series of studies in two neoplastic cell lines, Caco2 (colon adenocarcinoma) and SKBR3 (breast adenocarcinoma), qualitatively evaluating the internalization of the particles, determining with immunofluorescence the number of γ-H2AX foci after irradiation with ionizing radiation (3 Gy) and evaluating the viability rate of both cell lines after treatment by means of an MTT assay. Nanoparticle internalization varied between cell lines, though they both showed higher internalization degrees for functionalized GNPs. The γ-H2AX foci counts for the different times analyzed showed remarkable differences between cell lines, although they were always significantly higher for functionalized GNPs in both lines. Regarding cell viability, in most cases a statistically significant decreasing tendency was observed when treated with GNPs, especially those that were functionalized. Our results led us to conclude that, while 50 nm GNPs induce a clear radiosensitizing effect, it is highly difficult to describe the magnitude of this effect as universal because of the heterogeneity found between cell lines. Radiation therapy is one of the most common treatments when fighting neoplasms and almost 60% of patients receiving anti-neoplasic treatment undergo radiation therapy , be it aWith the aim of either diminishing the appearance of undesired side effects or increasing the radiation effect in tumoral cells, radioprotective and radiosensitizing agents can be used. A radioprotective agent reduces the damage produced in healthy cells by limiting the action on DNA of reactive radicals such as reactive oxygen species (ROS), mainly generated by the interaction of ionizing radiations and water . A pioneAn important limitation of some radiosensitizing treatments can be attributed to the unspecific distribution of the radiosensitizer throughout the body. One strategy for overcoming this limitation is the use of micro- or nanoparticles as radiosensitizers because they can naturally accumulate in tumor tissues due to the enhanced permeability and retention effect resulting from the imperfect endothelium of blood vessels formed during tumoral angiogenesis . To incrThe effect of nanoparticles as radiosensitizers has been extensively reviewed ,14,15,162 conditions. Doubling times were determined by counting stained cells with trypan blue  and visualizing by optical microscopy with the help of a hemocytometer (Sigma-Aldrich). Doubling times were 68 h for Caco2 cells and 63 h for SKBR3 cells.In the present study, two different cell lines were used: Caco2 derived from colon adenocarcinoma cells  and SKBR3 derived from breast adenocarcinoma cells (ATCC). Both cell lines were cultured with Eagle’s modified enriched medium  and McCoy’s 5A medium (ATCC), respectively, at 37 °C and in 5% CO® 488  and Goat Anti-Mouse IgG H&L Alexa Fluor® 488 . First, cells were centrifuged and rinsed with 1 × PBS, then 35.000 cells were centrifuged using a Cytospin centrifuge  and fixed to a polysine slide by applying 2% paraformaldehyde onto the sample. After 10 min, the slides were rinsed in 1 × PBS, and then fixed cells were permeabilized using 1 × PBS/0.5% Triton-100 at 4 °C. After 15 min, slides were again rinsed with 1 × PBS before applying a blocking solution (1 × PBS/0.1% Tween20/2% FCS) for 30 min. Then, cells were incubated with the primary antibody overnight in a humid chamber at 4 °C. Once incubation was complete, the slides were washed thrice for 5 min in a 1 × PBS/0.1% Tween20 solution to eliminate a possible primary antibody excess, then the secondary antibody was applied, and the slides were incubated for 1 h in a humid chamber at room temperature. After that, the slides were washed in 1 × PBS/0.1% Tween20 solution as previously described. Finally, 5 μL of Vectashield Mounting Medium  with 4,6-diamidino-2-phenylindole (DAPI) as counterstain at a 1.5 μg·mL−1 concentration was applied.To ensure that both cell lines behaved as expected and expressed the specific receptors (EpCam and TfR), an immunofluorescence staining was carried out. The primary antibodies used were Rabbit Anti-EpCam  and Mouse Anti-TfR , and the secondary antibodies were Goat Anti-Rabbit IgG H&L Alexa Fluor−1 for 4 h. Then, cultures were centrifuged and treated with a hypotonic solution (KCl 0.30 M) for 10 min, then cells were fixed with Carnoy’s solution (1 acetic acid:3 ethanol) and dropped onto slides. For the in situ hybridization, slides were firstly treated with pepsin/HCl (Sigma-Aldrich) (50 μg·mL−1) for 10 min at room temperature, washed twice with 1 × PBS for 5 min, fixed with formaldehyde 4%–PBS1×, and washed again with PBS . After that, slides were dehydrated in ethanol series  and air-dried. Then, hybridization with FITC-labeled pancentromeric and Cy3-labeled pantelomeric PNA probes  was performed. Shortly after, 10 μL of hybridizing solution with both PNA probes, 0.6 μM of the pancentromeric probe, and 0.4 μM of the pantelomeric probe were applied to the slides and covered with a coverslip. Cells and probes were denatured for 3 min at 80 °C and hybridized for 2 h at room temperature using a Vysis HYBrite Hybridization System . After hybridization, slides were washed twice, 15 min each, in 70% formamide–PBS1× and three times, 5 min each, in Tris-buffered saline solution, 1 × TBS/5% Tween20 (both from Sigma-Aldrich). Slides were then dehydrated with ethanol series and dried out, and 20 μL of the previously described DAPI solution were applied as counterstain and slides were observed on an epifluorescence microscope .To know the genomic stability of the cell lines used in the present study, first we analyzed the karyotypes of one hundred cells for each cell line. Fluorescence in situ hybridization techniques using pancentromeric and pantelomeric PNA probes were used to establish the modal karyotypes and to check for the presence of unstable chromosome aberrations. For this purpose, exponentially growing cell cultures were treated with colcemid at a final concentration of 0.3 μg·mLIn the present study, three types of GNPs were used. Citric acid stabilized 50 nm GNPs , from now on, non-functionalized gold nanoparticles (NF-GNPs), commercially Tf functionalized gold nanoparticles (TfGNPs)  and anti-EpCam antibody functionalized gold particles (AntiEpCamGNPs) functionalized with a conjugation kit  according to the manufacturer’s protocol. To measure the ζ-potential, functionalized and non-functionalized GNPs were separately resuspended in water or in EMEM (anti-EpCamGNP) and McCoy’s 5A (TfGNP) culture mediums and sonicated for 5 min  to achieve a monodispersed sample. The ζ-potential was then measured with a Zetasizer Nano ZS .5 nanoparticles·mL−1. Nanoparticle internalization was evaluated 24 h after treatment of the cell cultures with a broadband confocal microscope  located at the Microscopy Service of the Universitat Autònoma de Barcelona. CellMask™ Deep Red  was used to stain cell membranes and Hoechst 33342  to stain cell nuclei. Staining was applied 5 min prior to observation. Due to their reflective nature, no additional staining was required to visualize GNPs.Cell cultures of both cell lines were treated with 50 nm NF-GNPs. Moreover, Caco2 and SKBR3 cell cultures were treated with AntiEpCamGNPs and TfGNPs, respectively. In all cases, functionalized and non-functionalized GNPs, diluted in 1 × PBS, were added to the cell cultures at a final concentration of 7 × 10137Cs irradiator  located at the Technical Unit for Radiation Protection of the Universitat Autònoma de Barcelona. Dose rate was 5.02 Gy·min−1 and the energy peak was 662 keV. Cell cultures were irradiated with gamma-rays 24 h after incubation in the absence or in the presence of nanoparticles (NF-GNPs for both cell lines and TfGNPs for SKBR3 cells and AntiEpCamGNPs for Caco2 cells). Irradiations were carried out at 3 Gy with a To evaluate kinetics of γ-H2AX foci, five different cultures for each cell line were used, one to determine the basal frequency and four at different postirradiation times: 30 min, 2, 4 and 24 h. At each time, three irradiations were carried out for each cell line, one without GNP treatment, another in the presence of NF-GNPs and another with functionalized GNPs . To detect γ-H2AX foci, an immunofluorescence staining was conducted following the same protocol described in the γ-H2AX foci analyses were carried out using an automated scanning fluorescence microscope system  and processed using the MetaCyte software, version 3.10.2,  coupled to a motorized z-stage Zeiss Axio Imager.Z2 microscope (MetaSystems) The images were captured using a 63_PlanApo objective. The foci signals in the selected nuclei were captured using the SpOr filter (red channel). All the SpOr signals were acquired as a z-stack with a total of 10 focal planes and a z-step size of 0.35 μm between planes. A unique classifier was used to count foci in about 2000 nuclei for each experimental condition.4 cells·mL−1 concentration on a 96-well plate and cultured for 48 h before being treated with NF-GNPs, functionalized GNPs , or not treated at all in the case of our control cells. Twenty-four hours after GNP treatment, the culture medium was changed and then cells were irradiated at a 3 Gy dose. To determine the impact of treatments on cell viability, a 3--2,5-diphenyltetrazolium bromide tetrazolium reduction assay (MTT cell viability assay) was conducted following standard procedures 24 h after irradiation . Viability was calculated in relation to that observed in non-treated cells. Three replicates were carried out for each experimental condition.Cells were seeded at 4 × 10p values lower than 0.05 were considered statistically significant.Statistical analysis was conducted with IBM SPSS Statistics 24. Normality was tested in all cases using a Kolmogorov–Smirnov test with the Lilliefors correction. Since all cases showed a non-normal distribution, the statistical test chosen to compare foci frequencies was the Mann–Whitney U test. Viability comparisons were carried out with ANOVA with Tukey’s multiple comparison test. As expected, both cell lines expressed the cell membrane receptors (EpCam for Caco2 and TfR for SKBR3) described in the literature , confirmThe cytogenetic analysis by pancentromeric and pantelomeric fluorescence in situ hybridization  was carrThese results indicate that instead of the variability in the number of chromosomes, the cell lines are stable regarding the formation of unstable structural chromosome aberrations.In A qualitative assessment of GNP internalization through confocal microscopy imaging allowed us to detect certain differences between the cell lines . As can p < 0.001).The cytogenetic characterization did not show structural chromosome aberrations, but due to the variability in the number of chromosomes, about 2000 cells were analyzed for each different postirradiation time and GNP treatment. Microscope images of γ-H2AX foci in Caco2 and SKBR3 cells are shown in p < 0.01). For SKBR3 , the difTo know if the presence of GNPs could have any effect on the rate of foci disappearance with postirradiation time, foci decay with postirradiation time has been adjusted to a one-phase decay function, obtaining the constant of the decay rate and the foci half-life estimation for each experimental condition . For Cacp < 0.05). Moreover, for both cell lines the decrease in viability was more pronounced for cells irradiated in the presence of functionalized GNPs but was only significant for Caco2 cells (p < 0.05). After irradiation, SKBR3 cells always showed significantly lower viabilities than Caco2 cells (p < 0.05). The sensitization enhancement ratio for viability was also higher than one for functionalized particles  ,35 can dThe clonogenic assay has been used to measure the radioinduced cell death or loss of reproductive potential, though it has limitations such as the need for cell differentiation and the intrinsic variations in survivability between different cell types ,49. On tIn our study, working with two different tumoral cell lines has helped us to achieve a better knowledge of the nanoparticles’ radiosensitizing effect in different cell models. First, to know if the cells used were genetically unstable, we evaluated the chromosome stability of both cell lines, and although the chromosome number was variable in both cell lines, no cells with chromosomes lacking telomere signals were observed, indicating no structural chromosome instability and hence no influence in the posterior foci counts. The radiosensitizing effect of GNPs is attributed, in part, to the increase in ROS production with irradiation, but in the absence of radiation, GNPs increase the intracellular ROS level ,25,57,58In our study, we chose nanoparticles of 50 nm in diameter because previous studies indicated an increased cell uptake for GNPs of 50 nm in diameter in relation to 14 nm and 74 nm  and it hAnother factor, different to nanoparticles properties, to consider in studies of GNP radiosensitizing effect, is the intrinsic uptake capacities and intrinsic radiosensitivity of the cell lines. Not all cell types show a similar nano- or microparticle uptake capacity. SKBR3 cells can phagocytose 3 µm polysilicon–chromium–gold microparticles , 3 µm poRegarding the effect of GNPs in SKBR3 and Caco2 cell cultures without irradiation, we observed that the presence of NF-GNPs or functionalized GNPs did not significantly change the basal frequency of γ-H2AX foci or the cell viability when compared to cells grown without GNPs. This does not mean that the presence of GNPs does not have any effect on cells, because a slight decrease in cell viability, though not statistically significant, was observed when GNPs  were present in the cell culture. This agrees with studies describing an increase in ROS by the presence of GNPs without irradiation ,25,57,58After irradiation, the γ-H2AX foci frequency was evaluated at four different postirradiation times . For Caco2 cells, significant increases in foci frequencies were observed for cells irradiated in the presence of AntiEpCamGNPs when compared with cells irradiated in the presence of NF-GNPs or in the absence of GNPs. For these cells, the sensitization enhancement ratios (SERs) for foci obtained for AntiEpCamGNPs when compared with NF-GNPs or with irradiation without GNPs were, in general, close to the values described in the literature . MorFinally, when foci frequencies of both cell lines are compared, in all experimental conditions lower frequencies were observed in SKBR3 cells. This could be related to an elevated resistance to oxidative stress, as described for cells from breast cancer patients . HoweverAlthough high Z metallic nanoparticles are currently the most used for radiosensitizing, other non-metallic nanoparticles are showing promising results. In a study by Yamaguchi et al. , siliconDespite the radiosensitizing effect of gold nanoparticles previously described , the resEven with the differences in the results of the present study between the two cell lines, gold nanoparticles remain an interesting radiosensitizing agent and a good candidate for future in vivo application in the treatment of certain types of tumors. Active targeting of GNPs has demonstrated its effectiveness to increase the genetic damage and to decrease the cell viability. This GNP radiosensitizer effect should be explored in further studies to find more effective radiation therapies against different tumor types.


Medicinal plants are regarded as a large source of phytochemicals that may have anticancer properties. This could lead to the development of innovative drugs or alternative therapy against cancer.Bulbine frutescens, Bulbine natalensis, Chlorophytum comosum, Kniphofia uvaria, and Tulbaghia violacea.This study was designed to determine the antioxidant and cytotoxicity effect of 5 selected indigenous South African medicinal plants namely; Phytochemical extracts namely; methanol, 50%, 100% ethanol, and water extracts were prepared from the root and shoot of the plants. The antioxidant effect of methanol extracts of the plant materials was performed using a DPPH assay. A preliminary cytotoxicity screening of the phytochemical extracts in the human colon (Caco-2), cervical (HeLa), and hepatocellular (HepG2) cell lines were determined followed by the half-maximal inhibitory concentration (IC50) using MTT assay.B. natalensis and B. frutescens (33.20% and 26.33% respectively) and shoot extract of K. uvaria (17.10%) showed the highest antioxidant. Out of the 5 plants, only 100% ethanol extract of C. comosum, K. uvaria, and T. violacea caused more than 80% cytotoxicity in HepG2 and Caco-2 cell lines. The shoot of B. frutescens (10.43 µg/ml), K. uvaria (23.0 µg/ml), and root of C. comosum (23.77 µg/ml) were the most active with the highest cytotoxicity.The methanol root extract of C. comosum, K. uvaria, and T. violacea possess significant cytotoxicity that is promising in developing alternative drugs against colon and liver cancers. Our results provided new pieces of evidence for antioxidant and cytotoxic activities of these plants which could be useful for developing new anticancer therapies. Cancer is a life-threatening disease that severely affects the human populationSouthern Africa has over 30000 species of higher plants with most of them possessing medicinal properties which are promising for further development and commercializationThese South African indigenous plant species come from different families such as Asphodelaceae, Alliaceae, Hypo xidaceae, Agavaceae and Asteraceae. For centuries, these indigenous medicinal plants have been adopted for primary health care and treatmentsSecondary metabolites that are derived from plants are being investigated for the development of new clinical drugs to treat cancerMedicinal plants commonly known as herbs, pharmacologically active plants remain the dominant form of medicine in most countries.These plants are regarded as a large source of therapeutic phytochemicals (phenolic and flavonoids) that may lead to the development of innovative drugs against cancerBulbine frutescens, Bulbine na talensis, Chlorophytum comosum, Kniphofia uvaria, and Tulbaghia violacea were selected and tested for antioxidant and cytotoxicity activities. These selected medicinal plants have been used as traditional medicines for decades against diseasesBulbine frutescens, Bulbine natalensis, and Chlorophytum comosum cytotoxicity activities in human colon (Caco-2) and cervical (HeLa) cancer cell lines. Similarly, more information is needed regarding cytotoxicity activities of Kniphofia uvaria and Tulbaghia violacea, particularly, against hepatocellular (HepG2) cancer cell lines. Hopefully, our results may provide new information for anticancer drug development from these plants, particularly, against the aforementioned cancer cell line.In this present study, 5 indigenous South African medicinal plants, namely; Bulbine frutescens, Bulbine natalensis, Chlorophytum comosum, Kniphofia uvaria, and Tulbaghia violacea were obtained from Shadowlands Wholesale Nursery at Kuilsriver, Cape Town, South Africa. Plants were transplanted in potting soil and kept in an environmentally controlled greenhouse for steady and healthy growth. After 12 weeks, the shoot and root of the plants were removed individually from all selected plants. The plant materials were washed thoroughly with distilled water to ensure the removal of all the dirt, air-dried at 28°C for 4–8 days and then oven-dried at 48°C to remove residual moisture content.The dried plant materials were blended to a fine powder and stored at room temperature. Extracts were prepared as per methods described by Sousa et al.2 humidified incubator . The cells were seeded at 1 x 105 cells/ml density in a 96-well plate and incubated at 37 °C for 24 hrs. The viability of cells was evaluated by direct observation under the EVOS XL core imaging system .The human Caco-2, HeLa, and HepG2 cancer cell lines were obtained from Biolabels Node, University of Western Cape, Cape Town, South Africa. The cell cultures were prepared as previously described by Matsushita et al.2.All cell lines were maintained in a humidified incubator at 37°C in an atmosphere of 5% COThe antioxidant activity of the selected plants was measured in terms of radical scavenging ability using the stable radical DPPH as previously described by Kedare and SinghThe mitochondrial activity of cells is measured using a spectrophotometer through the reduction of a yellow-coloured MTT salt to purple formazan crystals by the enzyme succinate tetrazolium reductase or succinate dehydrogenase, present only in metabolically active cells. The greater the purple colour of the reaction, the less the extent to which a particular compound (crude fraction) induces cell death in the specific reaction. The cytotoxicity of the plant extracts in the cell lines was measured by two rounds of screening procedures in succession using MTT assay as previously described by Liang et al.An extract showing more than 80% cell cytotoxicity on at least 1 human cancer cell line in the preliminary screening was put into the secondary screening to assay its dose-dependent cytotoxic effects on the human cancer cell line. The half-maximal cytotoxic concentration of the active compounds that caused 50% cell growth inhibition (IC50) was investigated was performed based on the method previously described by Liang et al. B. frutescens, B. natalensis, C. comosum, K. uvaria, and T. violacea was determined through the percentage of DDPH inhibition by the plant extracts in the assay. The antioxidant activity through DPPH inhibiton assay of the 900 µg/ml methanol plant extracts is shown in B. natalensis (33.20%) and B. frutescens (26.33%) were found to exhibit the highest antioxidant in comparison to the root extract of other plants. Both C. comosum (10.33%) and K. uvaria (9.60%) showed a low level of antioxidant index, although it was significantly higher (p<0.05) when compared with T. violacea extracts (5.06%). In the shoot extract, the result showed that the free radicals were inhibited the most by K. uvaria (17.10%) and C. comosum (15.13%) compared to T. violacea (8.43%), B. natalensis (8.16%), and B. frutescens (7.86%).The antioxidant of the root and shoot methanol extracts of Experiment I: Selection of cytotoxicity above 80% cell growth inhibition.B. frutescens, B. natalensis, C. comosum, K. uvaria and T. violacea in Caco-2, HeLa and HepG2 cell lines using MTT assay. It was observed that only 100% ethanol extract of 3 plants  caused more than 80% cytotoxicity against HepG2 and Caco-2 cell lines as shown in C. comosum (90.47%) and T. violacea (90.20%). It was only C. comosum (root) that inhibits growth (88.20%) of Caco-2 compared to other root plant extracts. For the100% ethanol (shoot) extracts, K. uvaria inhibits (85.43%) growth of Caco-2. In HepG2, T. violacea and K. uvaria caused high cytotoxic activity (85.43% and 84.43%) respectively, while that of B. frutescens (71.56%) and B. natalensis (72.87%) cytotoxicity was high but not above 80%. It was noted that none of 50% ethanol and aqueous extracts of the selected 5 plants showed above 50% cytotoxicity against the cell lines. Also, none of the plant material extracts resulted in a cytotoxic effect > 80% against the HeLa cell line. To further confirm the cytotoxicity of plant extracts showing more than 80% cytotoxicity in the cell lines, we assayed all the extracts against HepG2 which was most sensitive to the 100% ethanol plant extract to determine the dose-dependent half-maximal cytotoxic concentration and the most active plant extracts that caused 50% cell growth inhibition (IC50). The 100% ethanol extracts for all the plants showed a stronger cytotoxic effect against HepG2 cells except for B. natalensis. The 100% ethanol extracts for B. frutescens (shoot), K. uvaria (shoot) and C. comosum (root) were the most active with IC50 = 10.43 µg/ml, IC50 = 23.0 µg/ml and IC50 = 23.77 µg/ml respectively exhibiting the highest cytotoxicity as shown in B. frutescens (root) IC50 value was 32.72 µg/ml and it was the only 50% ethanol extract amongst all the plant extracts to exhibit cytotoxic effect with IC50 < 50 µg/ml. The shoot of T. violacea was shown to have significant cytotoxicity with IC50 < 50 µg/mlA preliminary assay was performed to evaluate the cytotoxicity effect above 80% cell growth inhibition of 50%, 100% ethanol, and aqueous extracts of the roots and shoots of Bulbine frutescens and Bulbine natalensis showed high antioxidant ativities compared to the extracts from the shoot of both plants and the root and shoot extracts of T. violacea with low antioxidants. This possibly means the root part of the Bulbine sp contains sensitive phytochemicals capable of scavenging free radicals. A previous study reported that B. frutescens contain secondary metabolites such as quinones and glycoside which are potential anticancer agentC. comosum showed high antioxidant activity. In contrast, a previous study reported the root methanolic extract of C. comosum exhibited a strong and significant antioxidant activityC. comosum root extract in comparison to the ethanol and aqueous leave extracts. The concentration of the methanol solvents could affect the phenolic content which is a strong antioxidant agent in plant extractsB. frutescens (root and shoot), K. uvaria (shoot), and C. comosum (root) were confirmed the most active plant extracts showing the great cytotoxic effect (> 80%) and the IC50 values were between 10.0 – 35.0 µg/ml in HepG2. However, we noticed that T. violacea recorded high cytotoxicity with IC50 = 45.61 µg/ml. 16reported that methanol extract of B. frutescens induced reactive oxygen species (ROS) by 7%n cancer cell lines when compared to the untreated cells. Although, cancer cells may develop defense mechanisms against antioxidants and anti-proliferative agents which counters the effect of elevated ROS. Excessive production of ROS and potential changes in mitochondrial membrane is associated with the initiation of cell deathB. frutescens methanol and hexane extracts induced cell cycle arrest, ROS production, apoptosis, and mitochondria membrane potential disruption in breast cancer cellsBulbine sp induced potent cytotoxicity between 70 – 72% with B. frutescens IC50 value < 50 µg/ml compared to B. natalensis > 50 µg/ml. B. natalensis and B. frutescens induced a cytotoxic effect in the human laryngeal carcinoma cell line, however, the expression of caspase-3 which indicates apoptotic pathway was induced in shoot and leaf ethanol extracts except for the root of B. frutescens. Our study corroborates with previous studies which showed root extracts of C. comosum possess anti-proliferative activity against many human cancer cell lines. C. comosum. Saponins isolated from roots of C. comosum exhibited cytotoxic and antitumour promoter activity through induced apoptosis in selected cancer cell linesK. uvaria. The shoot extract of K. uvaria presented the highest antioxidant value compared to the other plants while the root extracts showed moderate antioxidant activity. K. uvaria is one of the most traditional medicinal herbs used for antimalaria and anti-inflammation but so far there is no research on the bioactivities related to its chemical components. We noticed there is little or no literature report on K. uvaria, however, K. foliosa through its derivative compounds, knipholone and knipholone anthrone demonstrated poor and potent antioxidant activity respectivelyK. foliosa contain anthraquinones, flavonoids, and alkaloids which have anticancer activitiesT. violacea induced a high cytotoxic effect in HepG2 and Caco-2 when compared to HeLa cell lines. T. violacea inhibited the growth of HeLa and HT-29 (colon cancer) cell lines. This was similar to our result which also showed a moderate degree of inhibition in both HeLa and Caco-2 cell lines. However, our study showed that the inhibition was high in the Caco-2 compared to HeLa as opposed 13 report showing high inhibition in HeLa in comparison to HT-2922. This could plausibly be the effect of the different solvent types used to extract the phytochemicals. Phytochemical compounds present in different parts of T. violacea may vary and these include tannins, terpenoids, flavonoids, saponins, proteins, steroids, cardiac glycosides, phenols and coumarins41In this present study, we evaluated the antioxidant effect of the phytochemicals that scavenged free radicals in the DPPH assay. The root methanolic extract of T. violacea exhibited apoptotic effect, although through a compound moiety-methyl-α-D-glucopyranoside which serves as a potential anticancer agent by interfering with hexokinase to inhibit the induction of ROST. violacea in HepG2 compared to other plants or cell lines could suggest that cytotoxicity was induced by an apoptotic pathway that could be caspase-3 dependentT. violacea extracts and cell lines to text its cytotoxicity is an important factor to always consider for future studiesWe noticed the aqueous extract has little or no cytotoxic effect on the selected cell lines. Previous studies showed that the aqueous extract from B. natalensis, C. comosum, K. uvaria and T. violacea against the selected human cancer cell lines implies the potential therapeutic source which can be utilized towards the development of new anticancer drugs. It is evident from this study that C. comosum, K. uvaria, and T. violacea possess significant cytotoxicity that is promising in developing alternative drugs against colon (Caco-2) and liver (HepG2) cancers. This study provides new insight into the potential bioactive properties, most importantly the antioxidant activity of K. uvaria. In general, different cell lines respond differently to the same compound. So it remains unclear whether similar results would emerge if other cell lines were used alongside the Caco-2, HeLa, and Hep-2 cell lines, hence further studies will be needed to unveil it. The report of Singh and ReddyBulbine spp. fractions that could. be used to further validate their findings. It is believed that our study has provided more information in addition to the existing data regarding the cytotoxicity of these plants. Phytochemical screening of the fractions may be required to identify the cytotoxic compounds in the fractions before this result can be extrapolated on animal models to study the in vivo effects. This ethnomedicinal knowledge could enhance the efficacy of the already existing or little ethnomedicinal use of these plants.The invitro antioxidant and cytotoxicity activities of B. frutescens, It is important to mention that further studies are needed to investigate the possible specific targets involved in the cytotoxic pathway of each plant extracts using molecular techniques.


Dysregulation of the epigenome due to alterations in chromatin modifier proteins commonly contribute to malignant transformation. To interrogate the roles of epigenetic modifiers in cancer cells, we generated an epigenome-wide CRISPR-Cas9 knockout library (EPIKOL) that targets a wide-range of epigenetic modifiers and their cofactors. We conducted eight screens in two different cancer types and showed that EPIKOL performs with high efficiency in terms of sgRNA distribution and depletion of essential genes. We discovered novel epigenetic modifiers that regulate triple-negative breast cancer (TNBC) and prostate cancer cell fitness. We confirmed the growth-regulatory functions of individual candidates, including SS18L2 and members of the NSL complex  in TNBC cells. Overall, we show that EPIKOL, a focused sgRNA library targeting ~800 genes, can reveal epigenetic modifiers that are essential for cancer cell fitness under in vitro and in vivo conditions and enable the identification of novel anti-cancer targets. Due to its comprehensive epigenome-wide targets and relatively high number of sgRNAs per gene, EPIKOL will facilitate studies examining functional roles of epigenetic modifiers in a wide range of contexts, such as screens in primary cells, patient-derived xenografts as well as in vivo models. Epigenetic modifications regulate gene expression and are altered by developmental and environmental cues . Strict CRISPR/Cas9 technology is a fast, effective, and easy-to-use genome engineering method and has drastically accelerated functional genomics research . Its simEpigenetic Knockout Library (EPIKOL), which targets a broader range of epigenetic modifiers and consists of more sgRNAs for each gene when compared to previously published libraries . Al. Al34]. To uncover epigenetic modifiers important for cancer cell fitness, we conducted negative selection (drop-out) screens using EPIKOL. Three different triple negative breast cancer (TNBC) cell lines MDA-MB-231, SUM149PT and SUM159PT were screened in addition to non-malignant human mammary epithelium cells (HMLE) . Similarp-values, log fold changes and gene rankings in different screens. 2 sgRNAs per gene were cloned individually into lentiGuide-puro vector and a dual-color competition assay was performed in all TNBC cell lines  is the catalytic member of the complex and acetylates histone lysine residues [SS18 gene, which is associated with chromosomal translocation characteristics of synovial sarcoma. However, the exact role of SS18L2 in synovial sarcoma or any other cancer is not known [To further delineate the effects of novel epigenetic modifiers that regulate cell fitness, four of the TNBC specific genes  were selected based on their strong depletion scores in MDA-MB-231 Fig. . Three oresidues . SS18L2 ot known . NSL comTo identify the mechanism through which cancer cell fitness is reduced, we first investigated whether knockout of the hit genes result in apoptosis. Extensive cleavage of PARP was observed with all four gene knockouts, indicating induction of apoptosis Fig. . SimilarTo assess the performance of EPIKOL in vivo, we performed an in vivo screen by using Firefly Luciferase (Fluc) expressing MDA-MB-231-Cas9 cell line. Following transduction and puromycin selection, cells were subcutaneously injected into Nude mice, and tumors were collected at weeks 2 and 4 post-implantation Fig. . Gene-leTo gain a mechanistic understanding for the essential role of SS18L2 in TNBC cells, we analyzed the transcriptomic changes caused by the knockout of SS18L2. We performed RNA sequencing comparing MDA-MB-231-Cas9 cells expressing control and SS18L2 sgRNAs. SS18L2 knockout resulted in 1283 upregulated and 255 downregulated genes Fig. . AverageIn this study, we present a focused epigenetic knockout library (EPIKOL) that can be utilized to investigate chromatin-based vulnerabilities in different biological contexts. We performed eight in vitro screens in two cancer types and identified novel chromatin modifiers that regulate prostate and triple-negative breast cancer cell fitness. In contrast to most currently available epigenome-focused libraries, which only target chromatin modifiers such as writers, readers, and erasers –42, EPIKAvailability of EPIKOL in LentiCRISPRv2 backbone might expedite the screening process by eliminating the need for prior Cas9 introduction especially in patient-derived xenograft models and primary cell lines, in which the culturing time of the material is limited. In such cases, smaller library size will also reduce the amount of initial material required to maintain the complexity. Another advantage of EPIKOL is the presence of sgRNAs targeting context-specific control genes from different families such as nuclear receptors, transporters and EMT-related proteins. For example, Androgen Receptor (AR) targeting sgRNAs were significantly depleted in AR-dependent prostate cancer cell lines LNCaP and 22Rv1 while no change was observed in AR-negative cell line DU145 and TNBC cell lines suggesting that EPIKOL can distinguish tissue or cell line specific hits.PRMT family, exosome complex and MLL complexes, highlighting the role of these epigenetic complexes as pan-cancer essential epigenetic modifiers. Some of these were previously identified as common essential genes in Cancer Dependency Map (DepMap) based on their significant depletion in almost 750 different cancer cell lines [ASH2L as a common essential gene in both cancer types through EPIKOL screens, but it was not classified as a common essential gene in previously performed screens. EPIKOL screen can therefore identify pan-cancer epigenetic vulnerabilities as well as cell line and cancer-specific ones. To test the performance of EPIKOL in vivo, we performed a screen using MDA-MB-231-Cas9-Fluc cells at two different timepoints. As expected, overall depletion scores and number of depleted positive control genes increased from week 2 to week 4. 9 out of 10 commonly depleted genes from both timepoints of the in vivo screen were shared with the TNBC in vitro screens. TFDP1 was identified as an in vivo specific cell fitness gene. These results indicate that EPIKOL efficiently works in vivo and can recapitulate the findings of in vitro screens.From drop-out screens in multiple cell lines, we identified novel epigenetic modifiers for cancer cell fitness as well as the previously studied ones such as PRMT5, HDAC3, FOXA1 and LSD1 , 59, 60.ll lines . NotablyWe identified a group of epigenetic modifiers that belong to NSL complex as TNBC-specific cell fitness genes. Core members KANSL2 and KANSL3 were depleted in three different TNBC cell lines but they did not show significant effects in HMLE cells. Importantly, KAT8, the catalytic subunit of the NSL complex, was also a common hit in MDA-MB-231 and SUM149PT cell lines. We showed that TNBC cells were dependent on NSL complex using several functional assays. NSL complex was thought to regulate H4K16Ac which is necessary for chromatin relaxation and present at active enhancers and promoters , 71. HowSS18L2 is a homolog of SS18, which is found in ATP-dependent chromatin remodeling complex BAF (SWI/SNF) and an uncharacterized gene [SS18L2. Knockout of SS18L2 markedly impaired tumor growth confirming that SS18L2 is required for TNBC cells in vivo. Transcriptomic analyses showed that SS18L2 regulates cell cycle-related pathways and genes that function during the late G2/M phase. For example, the DREAM complex activates late G2/M genes through its interactions with MYBL2 and FOXM1, both of which are downregulated upon SS18L2 suppression [In addition to the NSL complex, we observed SS18L2 to be significantly depleted in all TNBC EPIKOL screens. Competition assays confirmed the strong effect of SS18L2 knockout on TNBC cell survival. zed gene . Togethezed gene . The rolpression , 79. In pression . In the pression –42.In conclusion, we generated and validated a focused epigenetic sgRNA library that enables identification of critical epigenetic modifiers both in vitro and in vivo. Epigenetic modifying enzymes are promising therapeutic targets as they regulate numerous critical cellular responses including cell growth, metastasis, apoptosis and others. The relatively small library size both allows for loss-of-function screens where cell numbers are limited, and also provides a focused perspective for hit prioritization. EPIKOL is therefore a robust functional genomics platform to interrogate chromatin modifiers and can guide the discovery of cell-type specific epigenetic vulnerabilities of cancers.Co-author agreement documentSupplementary informationFULL LENGTH WESTERN BLOT IMAGESSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Reproducibility checklist file