Here, we describe a patient with post‐transplant myelofibrosis with chronic graft‐versus‐host disease (GVHD), who showed successful molecular remission with ropeginterferon with 100% donor chimerism without any flare up of GVHD. He was initially diagnosed with polycythemia vera (PV) which progressed to myelofibrosis after 6 years. The MYSEC (Myelofibrosis Secondary to PV and ET‐Prognostic Model) and MTSS (myelofibrosis transplant scoring system) scores were 13.1 and 4, respectively, and the patient was in intermediate risk group. He underwent an allogenic stem cell transplant; however, his disease gradually progressed and was administered two donor lymphocyte infusions with minimal response. A second allogeneic transplant was performed, which led to a persistent molecular remission for more than a decade, although he developed chronic skin graft‐versus‐host disease (GVHD). The JAK2 V617F levels started to increase 10 years post‐transplant with ongoing chronic GVHD and a corresponding decrease in donor chimerism levels. He was administered ropeginterferon, which led to a decrease in JAK 2617F to undetectable levels. A graft versus myelofibrosis effect was attained with reversal to 100% donor chimerism, and he has since maintained a molecular remission with undetectable JAK 2617F levels. Chronic GVHD made him ineligible for donor lymphocyte infusions later. Thus, ropeginterferon was successful in inducing graft versus myelofibrosis effect, leading to a molecular response with no flare up of GVHD. The use of ropeginterferon needs to be further evaluated in larger cohorts of post‐transplant myelofibrosis patients. JAK2 level and chimerism after ropeginterferon treatment for relapsed myelofibrosis after allogeneic stem cell transplantation. Various strategies have been tried in different clinical settings to achieve a GVL effect. Interferons have been in use for a long time in different malignancies; however, its side effects have led to decreased compliance over time. However, the availability of newer pegylated versions has made its use more convenient.Here we discuss a case of relapsed post‐transplant secondary myelofibrosis, with chronic GVHD, successfully treated with ropeginterferon. The patient achieved a graft versus myelofibrosis effect with 100% reversal to donor cell chimerism and complete molecular remission without flare up of GVHD after using ropeginterferon.248‐year‐old man initially presented to our institute in the year 2000 with a persistently raised hemoglobin (Hb) and hematocrit values for few months.9/L. Serum LDH (lactate dehydrogenase) was normal.Clinical examination revealed nontender mild hepatosplenomegaly. The peripheral blood counts revealed the following: Hb 23.5 g/L; hematocrit, 0.69; white blood cell count (WBC), 10.8 × 10 *9/L; neutrophils, 79%; lymphocytes, 10%; eosinophils, 2%; basophils, 1%; monocytes, 8%; platelets, 272 × 10Abdomen ultrasound revealed homogenous splenomegaly with a spleen size of 17 cm × 5.9 cm. The vertical dimension of the liver in the midclavicular line was 17.2 cm. There was no evidence of any lymphadenopathy.Bone marrow evaluation revealed a markedly hypercellular marrow, with pan myeloid hyperplasia and dominating hematopoietic hyperplasia. Overall findings were consistent with a diagnosis of polycythemia vera rubra.Cytogenetics revealed trisomy 1 and the patient was positive for JAK‐2 V617 F mutation. He was initially prescribed hydroxyurea and underwent venesections, which he well tolerated.Six years after the initial diagnosis of PV, in 2006, he presented with generalized worsening tiredness, night sweats, and weight loss of 3–4 Kg in the last few months. Clinically, his splenomegaly had progressed, and the spleen measured 15 cm below the left costal margin, almost pressing onto the left kidney. This was accompanied by an increase in bone marrow fibrosis. Thus, his disease had progressed to post PV‐myelofibrosis.9/L; platelets 487 × 109/L; LDH, 609 U/L.The MYSEC score (Myelofibrosis Secondary to PV and ET‐Prognostic Model) of 13.1 . Put him in the intermediate 1 group, but with worsening splenomegaly and B symptoms.Blood reports showed the following: Hb, 15.7 g/L; WBC, 24.0 × 10In 2006, he underwent his first HLA (human leucocyte antigen) unrelated (9/10 HLA matched) donor stem cell transplant.The conditioning regimen used was fludarabine, busulfan, and antithymocyte globulin (ATG) according to EBMT protocol.However, he had residual disease, which gradually progressed. A year later, in 2007, he underwent two donor lymphocyte infusions (DLI) 2 months apart with 1 × 10*7 CD3+ cells/kg; however, this produced no significant response as well as no GVHD.In 2008, he underwent a second HLA matched unrelated alternative donor transplant after conditioning with treosulfan, fludarabine, and ATG. This time he went into persistent molecular remission and remained in remission for a long time.However, simultaneously he developed acute gut GVHD, which initially responded to steroids. This later developed into chronic GVHD of gut and skin, and he underwent extra corporeal photopheresis (ECP) to which he partially responded.He, however, continued to be in persistent complete remission. His JAK2 V617F levels were undetectable, and he maintained a full donor chimerism.His disease remained in control for more than a decade, until in 2019 when his chimerism studies revealed a slight downward trend to 98.9% along with chronic moderate skin GVHD. The JAK2 V617F levels kept increasing correspondingly and increased up to 4.13 from being undetectable. Donor lymphocyte infusion was not indicated due to persistent chronic GVHD.He was administered ropeginterferon 250 ug subcutaneously once every 2 weeks. He tolerated this well without any significant side effects for almost a year (September 2019–November 2020) and then discontinued it. He did not demonstrate any major autoimmune side effects, depression, any other significant cytopenia, or worsening of GVHD. Serial studies of both chimerism and JAK2 V617F were done. His chimerism studies gradually improved with the present study showing full chimerism. His JAK2 V617F levels became undetectable in January 2021.Figure At present, the patient is not on interferon and is clinically asymptomatic with stable blood reports.This case shows that interferon can produce a graft versus myelofibrosis effect in patients with relapsed myelofibrosis after allogeneic stem cell transplantation without worsening GVHD.3Interferons were introduced into usage a long time ago due to their innate ability to produce resistance to viral replication. Type I and 2 interferons act on human interferon alpha receptor chains and activate signaling through the JAK/STAT pathway. Interferons can also enhance natural killer and T cells, macrophage activity, and enhance expression of tumor associated and major histocompatibility complex (MHC) antigens.Interferon has been used in myeloproliferative neoplasms for a long time. Its use in polycythaemia vera (PV) and essential thrombocytosis (ET) has led to a decrease in mutant alleles of JAK2 V617F and calreticulin (CARL) and this has led to a renewed interest in expanding its usage. Interferon can suppress clonal hematopoiesis in essential thrombocytosis and polycythaemia vera. Prior to tyrosine kinase inhibitors, interferons have been used in chronic myeloid leukemia (CML) and has induced cytogenetic remission.The PEGINVERA study also demonstrates the effectiveness of ropeginterferon in achieving 80% complete hematological and molecular response in patients with long‐term usage.Molecular landscape consists of JAK2 V617F, CARL, MPL, or in their absence other clonal driver mutations .Till now, allogenic stem cell transplant remains the only potential curative treatment of myelofibrosis. This case described here is of post‐transplant secondary myelofibrosis, where the patient had a molecular relapse with ongoing chronic skin GVHD. He was administered ropeginterferon which was tolerated well without any flare up of his GVHD. Serial monitoring of his JAK2 V617F levels and chimerism studies revealed an excellent sustained response to treatment. He was initially diagnosed with polycythaemia vera, JAK2 V617F positive, which progressed to secondary myelofibrosis. He had minimal response to the first allogeneic stem cell transplant and donor lymphocyte infusions was administered. The disease, however, progressed, and he proceeded on to a second allogeneic transplant. He gradually developed acute followed by chronic skin and gut GVHD for which he was administered steroids and received ECP , and he maintained his remission for a long time.A follow up after 11 years revealed a drop in chimerism and a rise in JAK2 V617F levels. However, he had ongoing chronic skin GVHD. He was administered ropeginterferon at a dose of 250 ug subcutaneously every fortnight, which continued for a year. This led to a gradual rise in chimerism and decrease in JAK2 V617F levels. His last reports (February 2022) revealed chimerism of 99.9%, and JAK2 levels of 0.006. Interestingly, his skin GVHD also responded well and did not show any flare up.JAK2 V617F allele frequency; however, this effect in advanced myelofibrosis, if any, is small. The possibility of the direct tumor effect of interferon leading to the remission in our patients cannot be ruled out. However, as seen in patients with relapsed CML after allograft, the major effect of interferon postallograft is to increase HLA restricted antigen presentation, which allows better donor T‐cell mediated graft versus leukemia effect.It has been observed that better responses were seen in non‐T depleted transplants highlighting the importance of donor T cells to produce a DLI effect in controlling the malignancy. In PV, interferon is known to cause a reduction in We have used the term graft versus myelofibrosis effect here as our patient is a case of advanced myelofibrosis.As reported by Steegman et al, interferon has produced durable, complete cytogenic response with acceptable toxicity in relapsed patients with postallogenic transplant CML.Similar studies have also shown that interferon (ropeginterferon) is quite effective in producing cytogenetic remission in relapse post‐transplant allogeneic transplant patients of CML.This emphasizes the potential of interferons in immunomodulating effect and maintaining a sustained remission in myeloproliferative neoplasms. This line of treatment can be considered in patients with persistent residual disease or relapse after allogenic stem cell transplant to increase a graft versus myelofibrosis/leukemia effect, especially if donor lymphocyte infusions are contraindicated due to GVHD.4Interferons have been in use for a long time. However, due to the side effects of the initial preparation and the access to newer molecules its use has transiently decreased. However, with the newer pegylated preparations, there has been an increase in its use in hematological malignancies. With careful considerations to the known side effects, it can be used in patients with relapsed myelofibrosis after allogeneic stem cell transplantation when DLI is practically not possible.The use of interferon in producing graft versus myelofibrosis needs to be explored in a larger cohort of myelofibrosis patients relapsing after allogeneic stem cell transplant.Barnali Srivastava: Conceptualization; data curation; formal analysis; project administration; resources; visualization; writing – original draft; writing – review and editing. Christine Wolschke: Conceptualization; data curation; methodology; resources. Nico Gagelmann: Conceptualization; data curation; formal analysis; resources. Anita Badbaran: Conceptualization; data curation; formal analysis; resources. Nicolaus Kröger: Conceptualization; data curation; formal analysis; project administration; resources; software; supervision; visualization.The authors declare that they have no conflict of interest.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.The new aspect shown in this study is the efficient use of ropeginterferon in inducing a 100% chimerism in relapsed post‐transplant myelofibrosis. The central finding of the study is the induction of graft versus myelofibrosis effect with ropeginterferon and attaining a successful molecular remission in relapsed post‐transplant myelofibrosis cases ineligible for donor lymphocyte infusions. The specific clinical relevance of the study is considering the use of ropeginterferon to produce molecular remissions and induce 100% chimerism in patients with relapsed post‐transplant myelofibrosis.


Background: One of the most significant challenges impeding cancer treatment effectiveness is drug resistance. Combining evolutionary understanding with drug resistance can pave the way for the identification of second-line drug options that can overcome drug resistance. Although capecitabine and irinotecan are commonly used therapeutic agents in the treatment of CRC patients, resistance to these agents is common. The underlying clonal dynamics of resistance to these agents using high-resolution barcode technology and identification of effective second-line drugs in this context remain unclear.Methods and materials: Caco-2 and HT-29 cell lines were barcoded, and then capecitabine and irinotecan resistant derivatives of these cell lines were established. The frequencies of barcodes from resistant cell lines and harvested medium, longitudinally, were determined. Collateral drug sensitivity testing was carried out on resistant Caco-2 and HT-29 cell lines using single agents or drug combinations. The SyngeryFinder tool was used to analyse drug combination testing.Results: In Caco-2 and HT-29 cell lines, barcode frequency measurements revealed clonal dynamics of capecitabine and irinotecan formed by both pre-existing and de novo barcodes, indicating the presence of polyclonal drug resistance. The temporal dynamics of clonal evolution in Caco-2 and HT-29 cell lines were demonstrated by longitudinal analysis of pre-existing and de novo barcodes from harvested medium. In Caco-2 and HT-29 cell lines, collateral drug sensitivity revealed a number of drugs that were effective alone and in combination.Conclusion: The use of barcoding technology reveals the clonal dynamics of chemotherapy-induced drug resistance not only from harvested cell populations, but also from longitudinal sampling throughout the course of clonal evolution. Second-line drugs that sensitize drug-resistant CRC cell lines are identified through collateral drug testing. Cancer is a global health issue that can affect people of all ages and genders, and it is responsible for the world’s second-highest disease-related death rate . Colorecde novo drug resistance  supplemented with 20% (v/v) Fetal Bovine Serum (FBS) , 1% (v/v) Penicilin-Sreptomycin , 1% (v/v) L-Glutamine , 1% (v/v) non-essential amino acids . HT-29 cell line was maintained in DMEM High Glucose Medium  supplemented with 1% (v/v) Penicilin-Sreptomycin  and 1% (v/v) L-Glutamine . PCR-based method was used to confirm The CloneTracker™ lentiviral barcode library  was used for the barcoding of Caco-2 and HT-29 cell lines. Lentiviruses were generated in HEK293T cell line using barcode library, pCMV-VSVG , and Delta 874. LV . Lentiviral infection was performed using 0.8 μg/mL polybrene. Multiplicity of infection (M.O.I.) 0.1 was achieved during lentiviral titration experiments and puromycin concentrations of 12.5 μg/mL for Caco-2 and 1.5 μg/mL HT-29 cell lines were used.6 barcoded Caco-2 and HT-29 cell were thawed and seeded into 15 cm cell culture dishes. When the cells were 70%–80% confluent, 2 × 106 cells per dish were seeded into four different 15 cm dishes with 25 mL medium  and 2 × 106 cell pellets were stocked as initial cell population. For Caco-2 cell line, mediums in the dishes were changed twice a week with fresh mediums containing IC50 dose (4 months) and subsequently 2x IC50 dose (2 months) of capecitabine, for HT-29 cell line IC50 dose (6 months) of irinotecan. Caco-2 and HT-29 cell lines treated with DMSO were given the same amount of DMSO used in dissolving compounds as fresh medium.A previously frozen cryovial of 4 × 10Following the end points of 6 months for each cell line, harvested cell lines were faced to genomic DNA isolation using GeneJET Genomic DNA isolation kit . Similarly, collected medium was centrifuged at 1,200 rpm and the collected pellet was used for genomic DNA isolation. For library preparation, barcode amplicons were amplified according to manufacturer’s guidelines provided by the Cellecta. Barcode sequences composed of 14 bp and 30 bp length variable nucleotides were sequenced on MiSeq Platform  using 150 bp pair-end method.The Illumina MiSeq Platform provided the barcode sequencing results of drug resistant harvested cell populations, their initial and DMSO controls and medium samples FASTQ format. FASTQC was used to evaluate the reads, and those with Phred scores less than 20 were excluded from further investigation. Trimmomatic was used to trim the FASTQ reads (parameter MINLEN:147), and pair-end reads less than 147 bp were eliminated . TrimmomThe detected unique barcode counts were classified based on their frequencies in capecitabine-resistant Caco-2 and irinotecan-resistant HT-29 biological replicates  and their DMSO controls. To determine the phenotypes of the barcodes, the following growth rate formula was used .r=1TlogR represents the frequency of the barcode(s) in the replicate, f0 represents the maximum frequency of all barcodes in the corresponding DMSO control, and T represents the time (week) between the first drug treatment and the end of the experiment. Barcode counts had less than 2 in all resistant samples and DMSO control were excluded from the analysis. Frequency of all barcodes in the corresponding DMSO control was used as the f0 value to calculate the growth rate of the barcodes. According to the calculated growth rates, the barcodes have a positive growth rate, and those detected at least two replicates were classified as “pre-existing”, the barcodes have a positive growth rate, and those detected just in one replicate were classified as “de novo”; the remaining detected barcodes were classified as “sensitive”, which had a negative growth rate.Here, fThe used medium was changed with fresh medium including drugs twice a week during the establishment of capecitabine-resistant Caco-2 and irinotecan-resistant HT-29 cell lines, and the floating dead cells in the used mediums were collected as pellets with each medium change. The genomic DNA from these cell pellets at five equal intervals (once every 4 weeks) was isolated and barcode sequencing were performed to track the frequency changes of the resistant barcodes in the cell populations.3 cells/well), along with their DMSO control and initial cells. Cells were treated with drugs after 24 h. The following drugs were used: capecitabine , irinotecan , SN-38 , and Cetuximab . The MTT cell viability assay was performed after 72 h of drug treatment.Capecitabine-resistant Caco-2 and irinotecan-resistant HT-29 ells were seeded into 96 well plates (10 × 103 cells/well). After 24 h, capecitabine-resistant Caco-2 and its control cells were given SN-38/capecitabine and cetuximab/capecitabine drug combinations, while HT-29 and its control cells were given dabrafenib/irinotecan and capecitabine/irinotecan drug combinations. Capecitabine and irinotecan were used as background drugs in drug combination assays, and their doses remained stable as 2x IC50 and 1XIC50, respectively. Capecitabine-resistant and control Caco-2 cells were given four doses of SN-38 and cetuximab in combination with capecitabine; irinotecan-resistant and control HT-29 cells were treated with five doses of dabrafenib and capecitabine. The MTT cell viability assay was performed after 72 h of incubation. Cell viability based on dose response curves was calculated using control wells, and synergy scores of drug combinations using cell viability results were determined using SynergyFinder 2.0 software . Using this software, the scores between −10 and +10 showed additive effect, +10 and more as synergistic, and −10 and less as antagonistic.Capecitabine-resistant Caco-2 cells and irinotecan-resistant HT-29 cells, as well as their DMSO controls, were seeded into 96-well plates  at 570 nm wavelength. GraphPad Prism 8 was used to calculate cell proliferation rates based on absorbance values .10 × 105/well for Caco-2 and 4 × 105/well for HT-29) were seeded in a 24-well plate containing growth. After cells reached to approximately 80% of confluency, cells were treated with 2 μg/mL Mitomycin  for 2 h. Scratches were made using 200 μL pipette tip and cell were washed three times to remove the debris. During scratch assay, DMSO control Caco-2 and HT-29 cells were treated with DMSO and capecitabine-resistant Caco-2 and irinotecan-were treated with capecitabine and irinotecan respectively, while full fresh growth medium were added onto the initial control cells. Migration of cells was monitored every 4 hours where the images were taken using Nikon Eclipse Ti2e microscope. The area of closure was calculated using previously published ImageJ software plugin .Total RNA was isolated and purified by using the Invitrogen PureLink RNA Mini Kit  according to the manufacturer’s instructions. Total RNA was isolated from frozen 2 × 1023 VN (50 µM) (2 µL), ProtoScript II Reaction Mix 2X (10 µL), ProtoScript II Enzyme Mix 10 X (2 µL) and Nuclease-free water .Reverse transcription protocol was applied on 1 µg total RNA of each sample. The RNA samples were diluted with d T)Real-time PCR experiments were conducted by using CFX Real-Time PCR Systems . 5 μL samples of cDNAs in 15 µL PCR buffer GoTaq q PCR Master Mix . The following primers were used:ABCB1-Forward: ACAGAAAGCGAAGCAGTGGT.ABCB1-Reverse: ATGGTGGTCCGACCTTTTC.Tm: 60°CGAPDH-Forward: TGGTCACCAGGGCTGCTTTT.GAPDH-Reverse:ACTCCACGACGTACTCAGCG.Tm: 60°Cp-values less than 0.05 were considered statistically significant. All results were given as mean SEM.Every experiment had at least three biological replicates, as well as at least three technical replicates. GraphPad Prism 8 was used for dose response curve data analysis . The statistical significance was determined using the Student’s t-test, two-way Anova, and nonlinear regression. The cellular barcoding method was used to investigate the clonal dynamics in Caco-2 and HT-29 cell lines under capecitabine and irinotecan selection, respectively . To ensude novo within the system is critical. Because the lentiviral barcode library integrated into the Caco-2 and HT-29 cell lines contains consensus forward and reverse primer sequences, it could be possible to calculate changes in individual barcode frequencies after drug treatment in comparison to the control DMSO sample. Furthermore, if the same barcodes were selected in parallel drug resistant cell lines, there is pre-existing drug resistance; whereas barcodes detected with increased frequency in parallel replicates but not shared in any of them will suggest the presence of potentially de novo drug resistance  were found to be 10.17%, 14.81%, and 1.71%, respectively.  were detected as 49.29%, 48.90%, and 37.88%, respectively  . To bettAs before, we wanted to see if irinotecan-induced resistance in HT-29 replicate B had a cost of resistance and thus collateral sensitivity to second-line drugs. We first wanted to check if irinotecan resistance affected growth rate as a cost of resistance in the most irinotecan resistant HT-29 replicate B. The growth rate assay revealed that HT-29 replicate B grew slower than the initial and control DMSO HT-29 cell lines . ContrarNext, we wanted to see if combining capecitabine and dabrafenib with irinotecan would facilitate or improve collateral sensitivity in HT-29 cells that were resistant to irinotecan. The MTT cell viability assay showed the effect of collateral sensitivity for combination of capecitabine, and irinotecan as indicated by 8.7-fold decrease in IC50 value, suggesting the importance of this drug combination for collateral sensitivity in irinotecan-resistant HT-29 cells . Next, wde novo barcodes, both from harvested end points where cells became resistant to capecitabine and irinotecan, and via temporal evolutionary dynamics from monthly medium in a non-destructive way. The predominance of evolving tumour populations in different tumour types, which evolve in a branching and linear pattern, as well as the evolution of tumour populations in response to therapy, support the polyclonal drug resistance observed in this study  detected following 10 days of dabrafenib treatment  and immune cells. Given the important roles of CAFs in mitigating drug sensitivity and immune cells in immune evasion , identifis study , and henis study , which c


Bombyx mori, and possesses endogenous small interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) pathways. BmN-4 cells are latently infected withBombyx mori latent virus(BmLV), an RNA virus whose replication is strictly controlled by both siRNA and piRNA pathways. Knockdown or knockout of the core factors of these two small RNA pathways increases BmLV RNA amount, which in turn inhibits cell growth. Here, we used the known RNAi suppressor CrPV-1A to assess whether the BmN-4 cell line can be used for screening the suppressors of siRNA and piRNA pathways.The BmN-4 cell line originates from the ovaries of silkworm, Bombyx mori cell line BmN-4 is a well-known ovary-derived cell lineCultured cell lines are an important biological resource for producing recombinant proteins and evaluating gene function. The silkwormBombyx mori latent virus is a positive, single-stranded insect RNA virus that is closely related to plant maculaviruses. BmLV was first discovered in BmN-4 cells, and has been found to infect almost allB. mori-derived cultured cell linesIn this study, we tested whether BmN-4 cells can be used to assess the suppressor activity of foreign genes by measuring the expression levels of BmLV genes and observing the degree of inhibition of cell growth. We selected the cricket paralysis virus 1A protein (CrPV-1A) gene as the suppressor gene. CrPV-1A has been found to inhibit Ago2-dependent RNAi via blocking the initial target searching by Ago2-RISCCrPV-1Ainto the vector, pIZ/His-V5-g3, so that the cloned gene would be expressed under the control of theB. mori hsp90promotercoat protein(cp) andRNA-dependent RNA polymerase(rdrp)  , suggesrdrp) . Taken Cell lineBmN-4 cells Plasmid constructionA CrPV-1A fragment was amplified from pCold II-CrPV-1A (provided by Yukihide Tomari)Transfection and generation of stably transfected BmN-4 cells5cells per 35 mm dish) were transfected with 1 µg of pIZ/V5-His-g3-GFP or pIZ/V5-His-g3-GFP-CrPV-1A using FuGENE HD (Promega). Three days after transfection, zeocin  was added to the mediumTMcell imaging station (Life Technologies). Thereafter they were collected for RNA extraction.BmN-4 cells  and then subjected to reverse transcription with avian myeloblastosis virus reverse transcriptase and an oligo-dT primer (TaKaRa). RT-qPCR was performed using a KAPA SYBR FAST qPCR kit (Kapa Biosystems) and the specific primers. The expression values were calculated using the 2PCR primers for CrPV-1ACrPV1A-f: TACCGAGCTCGGATCatgtcttttcaacaaacaaacaacaacgcCrPV1A-r: GCCACTGTGCTGGATctagaaggctctgcattcatcattaccpqPCR primers for BmLVcoat-2F: TCCTCTCGCATTACTATTGGcoat-2R: ATGGAGCCTCTGATGACAACrdrpqPCR primers for BmLVrdrp-2F: TCTCTCATGAAATCAGCACCrdrp-2R: TCACGATATGGTTTGAGATGB. mori rp49qPCR primers forrp49-F: CCCAACATTGGTTACGGTTCrp49-R: GCTCTTTCCACGATCAGCTT


Chondroitin sulfate proteoglycan 4 (CSPG4), also known as high molecular weight-melanoma associated antigen, is expressed in melanoma but also other tumor entities and constitutes an attractive target for immunotherapeutic approaches. While recent preclinical reports focused on anti-CSPG4 chimeric antigen receptors (CAR), we here explore T-cell receptor (TCR)-based approaches targeting CSPG4.+ melanoma, glioblastoma and lung cancer cell lines were identified and, if negative, retrovirally transduced with HLA-C*07:01.The TCRs of two CSPG4-reactive T-cell clones (11C/73 and 2C/165) restricted by the highly prevalent HLA-C*07:01 allele were isolated and the respective αβTCR pairs were retrovirally expressed in CRISPR/Cas9-edited TCR-knockout T cells for functional testing. We also combined alpha and beta TCR chains derived from 11C/73 and 2C/165 in a cross-over fashion to assess for hemichain dominance. CSPG4+HLA-C*07:01+ target cells by the αβTCR retrieved from the parental T-cell clones and in part also by the cross-over TCR construct 2Cα-11Cβ. Despite high surface expression, the 11Cα-2Cβ combination, however, was not functional.Functional tests confirmed specific recognition of CSPG4+HLA-C*07:01+ cancer cells which warrants further preclinical and clinical evaluation of these TCRs.Collectively, 11C/73- and 2C/165-expressing T cells specifically and efficiently recognized CSPG4 Patient-derived cell lines were established with informed consent and in accordance with the local ethical guidelines. Melanoma lines were A375, provided by Krishnaraj Rajalingam , D03, D05, D10, D14, D17, D18, D22, D28, D41, provided by Chris W. Schmidt , Ma-Mel-66b, Ma-Mel-86b, Ma-Mel-86c, provided by Anette Paschen , MZ7-Mel#1, provided by Thomas Wölfel . Glioblastoma cell lines MZ-219-GBM, MZ-221-GBM, MZ-222-GBM, MZ-257-GBM, MZ-304-GBM, MZ-373-GBM, MZ-415-GBM, MZ-416-GBM, MZ-483-GBM and lung cancer cell lines LC-MZ-1, LC-MZ-6, LC-MZ-16 were provided by Sigrid Horn . Lung cancer cell line NW-BC-11 was provided by Elke Jäger . Renal cell cancer cell lines RCC 1170, RCC 1774, RCC 1795, RCC 1846, RCC 1851, RCC 1879, RCC 1940, RCC 1973, RCC FM-KOT, RCC PB5, RCC PB6, RCC PB7 were provided by Barbara Seliger . Phoenix-Ampho cells were maintained in DMEM  supplemented with 10% fetal calf serum , 1% Penicillin/Streptomycin, 1% L-Glutamine  and 25 mM HEPES buffer . All other cell lines were maintained in RPMI (Thermo Fisher Scientific) supplemented with 10% FCS and 1% Penicillin/Streptomycin. T cells were maintained in Panserin 413 medium  supplemented with 10% human serum  and 1% Penicillin/Streptomycin. All cells were kept at 37°C in a humidified atmosphere.2.2The T-cell clones 11C/73 and 2C/165 were weekly restimulated with irradiated (100 Gy) antigen-expressing tumor cells (Ma-Mel-86c) at a stimulator-to-T cell ratio of 1:10 in the presence of 250 IU/mL IL-2 .2.3+ or CD8+ T cells were isolated from PBMCs by positive selection using magnetic bead separation  and stimulated for 48 h with the anti-CD3 mAb OKT3 (30 ng/mL) (Milteny) in the presence of 600 IU/ml IL-2 (Novartis) and the irradiated (100 Gy) cells of the flow-through of the isolation procedure.PBMCs were isolated by Ficoll gradient centrifugation from buffy coats of healthy donors obtained from the blood bank of the University Medical Center of the Johannes-Gutenberg university Mainz. CD42.4+ or CD8+ T cells were washed twice with PBS and once with Opti-MEM (Thermo Fisher Scientific) and were subsequently resuspended in 100 µL supplemented Nucleofector Solution  per cuvette at a concentration of 4-5e6 T cells/100 µL. In parallel, RNPs targeting the TRAC (TCAGGGTTCTGGATATCTGT) and TRBC1/2  and anaGiven the high functional avidity and confirmed specificity of both T-cell clones, we next employed the 5’-RACE method  to ident3.2+ T cells can be redirected against CSPG4. Notably, our TCR-expression-constructs incorporated optimizations aimed to reduced mispairing of transgenic TCRs with the endogenous TCRs such as replacing the human constant regions with their murine counterparts . To further reduce any residual TCR mispairing, expression of the endogenous TCRs was abrogated by electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs) targeting the TRAC and TRBC1/2 loci. Following human TCR (hTCR) knockout, TCR-KO CD8+ T cells were retrovirally transduced with the cTCR expression constructs and were expanded by weekly stimulation with anti-CD3/CD28 beads in the presence of IL-2. Of note, in addition to transducing the parental αβTCR gene-pairs, we also combined the alpha and beta chains of the 11C/73- and 2C/165-TCRs in a cross-over fashion to assess for potential TCR hemichain dominance .The αβTCR sequences of both clonotypes were subsequently cloned into retroviral expression constructs shown in HLA-C*07:01 and two different amounts of CSPG4:1-562 cDNA. CD8+ T cells transduced with the 11Cαβ and 2Cαβ TCR constructs specifically recognized CSPG4:1-562 cDNA-transfected targets . We then tested the parental T-cell clones or CD8+ T cells expressing the indicated transgenic cTCR for recognition of our target panel in IFNγ-ELISpot assays. We observed high IFNγ spot counts with both T-cell clones against all cell lines tested with T-cell clone 2C/165 showing higher spot counts against D10, D41 and MZ-GBM-222 compared to T-cell clone 11C/73. Transgenic CD8+ T cells expressing the 11Cαβ- or 2Cαβ-cTCR elicited IFNγ spot formation above background levels when co-cultured with the endogenously HLA-C*07:01+ melanoma cell lines D10, D41, the glioblastoma cell line MZ-257-GBM/HLA-C*07:01 and the lung cancer cell line MZ-LC-16/HLA-C*07:01 . When expressed as chimerized TCR constructs in T cells both TCRs specifically recognized the CSPG4:554-562 peptide as well as endogenously processed CSPG4 with similar high functional avidity. CSPG4 has been previously investigated as an immunotherapeutic target for CAR T cells, among others in melanoma and glioblastoma –19, and TRAC, TRBC and PDCD1 loci by CRISPR/Cas9 was safe and feasible  interactions thereby increasing functional T-cell avidity clinical evaluation particularly in the context of melanoma and glioblastoma. Further studies may focus on the 2C/165 TCR due to its higher avidity that translated also into increased reactivity against HLA-C*07:01patients , 15, and antigen .The original contributions presented in the study are included in the article/Patient-derived cell lines were established with informed consent and in accordance with the local legislation and institutional requirements. Human blood products were acquired from the blood bank of the University Medical Center of the Johannes-Gutenberg university Mainz in accordance with the local legislation and institutional requirements.CW and KK designed the study, performed experiments and curated data. KK wrote the original draft. MFatho and EH performed ELISpot experiments and MFatho curated these data. SL, VL and MFatho derived the T-cell clones 11C/73 and 2C/165 from Ma-Mel-86 blood lymphocytes. MFaust generated one of the TCR expression constructs. AP established and provided the Ma-Mel-86 model. MT provided essential resources and contributed to the conceptualization of the study. TW acquired external funding and supervised the study. CW, MFatho, TW, and MT edited the draft manuscript. All authors contributed to the article and approved the submitted version.


A three-dimensional spheroid cell culture model resembles the architecture and cellular characteristics of solid tumors much more closely than the conventional monolayer cell cultures often applied in preclinical drug testing. In this study, we validate the efficacy of the RIST treatment protocol, a novel multimodal treatment regimen for high-risk neuroblastoma, in a neuroblastoma spheroid cell culture model characterized by an augmented neoplastic phenotype. The results also indicate the importance of the neuroblastoma spheroid model for preclinical drug testing in a rigorous, robust and efficient high-throughput format, which can be beneficial to identify more effective treatment options for children with high-risk neuroblastoma in the future.Background: The outcome for patients with high-risk neuroblastoma remains poor and novel treatment strategies are urgently needed. The RIST protocol represents a novel metronomic and multimodal treatment strategy for high-risk neuroblastoma combining molecular-targeted drugs as ‘pre-treatment’ with a conventional chemotherapy backbone, currently evaluated in a phase II clinical trial. For preclinical drug testing, cancer cell growth as spheroid compared to mo-nolayer cultures is of advantage since it reproduces a wide range of tumor characteristics, including the three-dimensional architecture and cancer stem cell (CSC) properties. The objective of this study was to establish a neuroblastoma spheroid model for the rigorous assessment of the RIST treatment protocol. Methods: Evaluation of CSC marker expression was performed by mRNA and protein analysis and spheroid viability by luminescence-based assays. Aberrant expression of RNA-binding protein La in neuroblastoma was assessed by tissue microarray analysis and patients’ data mining. Results: Spheroid cultures showed increased expression of a subgroup of CSC-like markers  and higher Thr389 phosphorylation of the neuroblastoma-associated RNA-binding protein La when compared to monolayer cultures. Molecular-targeted ‘pre-treatment’ of spheroids decreased neoplastic signaling and CSC marker expression. Conclusions: The RIST treatment protocol efficiently reduced the viability of neuroblastoma spheroids characterized by advanced CSC properties.  As the most common extracranial solid tumor in childhood , neuroblThe RIST therapy, a novel multimodal treatment design for high-risk NB, is based on metronomically combining molecular-targeted biologicals, mTOR inhibitor Rapamycin and tyrosine-kinase inhibitor Dasatinib, with a conventional well-established chemotherapeutic backbone, consisting of the topoisomerase inhibitor Irinotecan and alkylator Temozolomide . The mecThe majority of in vitro drug testing on cancer cells is performed on conventional two-dimensional (2D) cell cultures . NonetheCancer stem cells (CSCs) are known to have high tumorigenic potential, combining self-renewal, pluripotency and proliferating capacity with resistance to treatments such as conventional chemotherapy and radiation ,25,26,27The aim of this study was to assess the RIST treatment protocol in a neuroblastoma spheroid culture model. Optimal conditions for spheroid growth on 96-well ultra-low-attachment (ULA) plates for three MycN-amplified (MNA) (SK-N-BE(2), Kelly, IMR-32) and two MycN-non-amplified (MNN)  NB cell lines were established. The 3D cultures were defined by measuring the size and viability of spheroids, as well as the expression of three CSC-like markers . Our results demonstrated the effective inhibition of neoplastic signaling and reduced expression of a subgroup of CSC-like markers after molecular-targeted ‘pre-treatment’ in spheroids of all NB cell lines tested.2 atmosphere. The SK-N-SH, SK-N-AS and SK-N-BE(2) lines were cultured in 1:1 EMEM /Ham’s F12 , supplemented with 10% heat-inactivated fetal bovine serum , 1% penicillin/streptomycin  and 2 mM L-glutamine . The cell line IMR-32 was cultured in RPMI 1640  with 20% heat-inactivated FBS, 1% penicillin/streptomycin and 1% non-essential amino acids . The cell line Kelly was cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin and 2 mM L-glutamine. The cell line SK-N-MC was cultured in DMEM  supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, 1% MEM NEAA and 2 mM L-glutamine. All cell lines were regularly tested for mycoplasma contamination. For 3D cultures, ultra-low-attachment (ULA) 96-well plates with round bottoms  were used. FBS was substituted for 5% knock-out serum replacement  [Cell culture: Neuroblastoma and Ewing’s sarcoma cell lines were originally purchased from either the American Type Culture Collection, ATCC , or from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ (SK-N-BE(2), IMR-32, Kelly). Cells were cultivated at 37 °C in a 5% CO1815-01)  in SK-N-1815-01) ,35,36.Drug treatment: Temozolomide , SN-38, the active metabolite of Irinotecan , Dasatinib (#D-3307) and Rapamycin (#R-5000), both purchased from LC Laboratories, were dissolved in 100% dimethyl sulfoxide  as a vehicle and stored at −20 °C. All drugs were diluted in cell-line-appropriate media to the desired concentrations immediately before treatment and applied at the indicated time points. Appropriate DMSO concentrations <1% final concentration were used as a control. Drug concentrations for the combined treatments are listed in Table 2. A scheme of the experimental design to evaluate the impact of RIST treatment on spheroid viability is displayed in Figure 4.50 concentration, the CellTiter-Glo® 3D Cell Viability Assay , based on measuring an ATP-dependent luminescent signal, was performed according to the manufacturer’s instructions [® Plate Reader and analyzed with the CLARIOStar® MARS analysis software. Cell viability was calculated by normalizing the measured viability to DMSO (vehicle) controls. CellTox Green Dye  was applied according to the manufacturer’s instructions. Three replicates were used for each drug combination and experiments were independently repeated at least three times.Viability analysis: To determine the ICructions . The lumRNA isolation and cDNA synthesis: Total RNA from NB spheroids was isolated using the RNeasy Mini Kit , following the manufacturer’s instructions. For purifying the isolated RNA, DNase digestion with DNase I was performed using the RNAse-free DNase Set . Quantification and purity were measured with the ND spectrophotometer NanoDrop. Complementary DNA (cDNA) was reverse-transcribed using the LunaScript RT Supermix , according to the manufacturer’s instructions, with ‘No RT’ controls to verify the absence of genomic DNA in each template and therefore prevent quantification interference during qPCR.2qPCR Primer Assay (Qiagen).Quantitative PCR (qPCR): To test for CSC marker expression, qPCR was performed using the SYBR Green Master mix  on a thermocycler system . PCR product quantification was measured by the relative quantification method of Livak and Schmittgen , normaliImmunoblot analysis: Cells were lysed and protein was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The following antibodies were applied: 4E-BP1 , phospho-4E-BP1, MycN , p70S6 kinase , phospho-p70S6 kinase , Src kinase , phospho-Src , GAPDH , CXCR4 , BMI1 , NANOG , La protein (antibody 3B9 ) and phoImmunohistochemistry: The immunohistochemical staining was performed applying the monoclonal human La-specific 3B9 antibody, as described in detail in ,42, on at-test. p values < 0.05 were considered statistically significant. Three degrees of significance were determined: * < 0.05, ** < 0.01 and *** < 0.001.Statistical analysis: All experiments were performed at least three times. The results were stated as means of at least three technical replicates ± standard deviation (SD). For microscope image analysis, ImageJ 1.53a  was used. Statistical data analysis was performed on GraphPad Prism 9 using the unpaired Student’s To define optimal spheroid culture conditions for various NB cell lines, we seeded increasing cell numbers from 10 to 1000 cells per well of two MycN-non-amplified (MNN) NB cell lines (SK-N-SH and SK-N-AS) in round-bottom ULA 96-well plates and monitored spheroid growth over time by taking phase-contrast images a; we detIn addition, we monitored spheroid growth for three MycN-amplified (MNA) cell lines (SK-N-BE(2), IMR-32, Kelly) by plating again increasing cell numbers in round-bottom ULA 96-well plates and monitoring the phenotype and size of spheroids over time. After 7 days, SK-N-BE(2) and IMR-32 formed compact, round spheroids, whereas Kelly formed spheroids with less defined edges . FurtherTesting whether NB spheroids gained cancer stem cell (CSC) properties, we determined the mRNA expression levels of CSC-like markers in cells grown as spheroids in 3D culture compared 2D monolayer culture. Hence, for all five NB cell lines, we isolated RNA from both monolayer and spheroid cultures and quantified the mRNA expression of CSC markers CXCR4, NANOG and BMI1 by reverse transcription followed by quantitative PCR. In all NB cell lines tested, the CXCR4 mRNA expression was increased in spheroid compared to monolayer cultures. In MNN cell lines, CXCR4 mRNA expression was dramatically upregulated (10- to 18-fold), whereas, in MNA cell lines, the increase was 3- to 5-fold in spheroid cultures a. FurtheTaken together, these data indicate a strong increase in CXCR4 mRNA expression in NB spheroids compared to monolayer cultures for all five NB cell lines tested. Interestingly, the effect was noticeably pronounced in NB spheroid cultures of MNN when compared to MNA cell lines. Furthermore, NANOG and BMI1 mRNA expression was also, although to a lesser extent, augmented in spheroids compared to monolayer cultures in both MNN cell lines (SK-N-SH and SK-N-AS) and BMI1 mRNA expression in MNA cell line IMR-32.The novel multimodal RIST treatment protocol combines two molecular targeted drugs, the mTOR inhibitor Rapamycin and the tyrosine-kinase inhibitor Dasatinib, as a ‘pre-treatment’, with conventional chemotherapeutics, the topoisomerase inhibitor Irinotecan and the alkylating agent Temozolomide . Previou50) for single-drug treatment applying viability assays, followed by determining the synergistic effects of combination treatments by using the Chou–Talalay method [50 values applied for single-drug treatments [Here, we tested whether the R+D ‘pre-treatment’ affects the viability of NB spheroids. Therefore, we seeded NB cells in round-bottom ULA 96-well plates and treated them with R+D at the concentrations depicted in y method ,44,45. Deatments ,44.To test whether the R+D ‘pre-treatment’ affected the viability of NB spheroids, cells were seeded in round-bottom ULA 96-well plates according to seeding numbers, as depicted in To determine how R+D ‘pre-treatment’ with Rapamycin plus Dasatinib (R+D) affects the neoplastic signaling response, the mammalian Target of Rapamycin (mTOR) signaling pathway and phosphorylation of the Src kinase family were assessed in NB monolayer and spheroid cultures. Herein, we analyzed the impact of Rapamycin on the phosphorylation status of downstream targets of mTOR complex 1 (mTORC1), such as the phosphorylation of ribosomal protein S6 kinase (p70S6K) at threonine 389 (Thr389) and the phosphorylation of translation initiation factor 4E-BP at serine 65 (Ser65). Furthermore, changes in the phosphorylation status of Src kinase at tyrosine 416 (Tyr416) upon tyrosine-kinase inhibitor Dasatinib treatment were analyzed. We treated monolayer and spheroid cultures of the MNN cell line SK-N-AS and the MNA cell line SK-N-BE(2) with R+D or with vehicle (control) at the concentrations depicted in Our above-reported results  revealedNotably, in neuroblastoma cell lines SK-N-AS and SK-N-BE(2), the protein expression of CXCR4, BMI1 and NANOG was raised in spheroid compared to monolayer cultures, suggesting an augmented cancer stemness phenotype expressed under 3D culture conditions b. Most iTo test whether the RIST treatment affects the viability of NB spheroids, we seeded five different NB cell lines in round-bottom ULA 96-well plates and treated them on the same day with the combination of Rapamycin plus Dasatinib (R+D) at the concentrations depicted in The RNA-binding protein La is overexpressed in a variety of cancer entities and has been shown to play a critical role in cancer pathobiology . Immunohhttps://r2.amc.nl, dataset ID: GSE62564, accessed on 9 March 2023) revealed a significant correlation between high La mRNA expression and the reduced overall survival probability of neuroblastoma patients -induced epithelial to mesenchymal transition (EMT) and the gain of CSC properties in head and neck cancer cells . The TGFImmunoblot analysis revealed a slight increase in the Thr389 phosphorylation of La in MNA neuroblastoma cell lines ) when compared to MNN neuroblastoma  and Ewing‘s sarcoma (SK-N-MC) cell lines a. ComparNew treatment strategies are urgently needed for high-risk children with relapsed or treatment-refractory neuroblastoma. Targeting relevant signaling pathways—such as the neoplastic PI3K/Akt/mTOR pathway—revealed promising efficacy in monolayer NB cell cultures, and corresponding therapeutic agents were tested in various clinical trials . HoweverIn the present study, we established a spheroid cell culture model for neuroblastoma. Compared to monolayer cultures, NB spheroids were characterized not only by significantly higher expression of a subgroup of CSC markers, including CXCR4, NANOG and BMI1, but also increased T389 phosphorylation of cancer-associated RNA-binding protein La. Nevertheless, treatment with the multimodal RIST protocol efficiently inhibited neoplastic signaling, viability and CSC-like marker expression in NB spheroids. The results indicate the efficiency of the RIST treatment protocol and, in addition, emphasize the NB spheroid model as favorable compared to monolayer cultures for the preclinical testing of new drugs in a rigorous and efficient screening format, which can be beneficial to identify more effective treatment options for children with high-risk neuroblastoma in the future.MycN amplification is well established as an important predictor of poor outcomes in NB patients and defines 50% of the high-risk population . ConsequSeveral studies have indicated that elevated CSC marker expression is induced by spheroid growth ,22. CompChemokine-receptor 4, CXCR4, after binding its ligand, stomal-derived factor 1, SDF1 (CXCL12), is important for the homing of hematopoietic stem cells and establishing a stem cell niche within the bone marrow . In NB pThe pluripotency-maintaining transcription factor NANOG maintains a high self-renewal capacity of embryonic stem cells and is a CSC-like marker associated with poor prognosis in neuroblastoma ,29,33,59Our study demonstrates, for the first time, the aberrant overexpression of RNA-binding protein La in neuroblastoma. Furthermore, data mining revealed that high La mRNA expression correlates with poor overall survival in neuroblastoma patients. These findings are in agreement with several former reports demonstrating the aberrant overexpression of the RNA-binding protein La in a variety of cancer entities, including lung, cervical and head and neck cancer, as reviewed recently . Cancer-An accurate analysis of cell viability and susceptibility to the given treatment is necessary to assess its preclinical efficacy and guide the development of new therapeutics with high precision. To decrease the discrepancy between in vivo tumor tissue and in vitro cell cultures, spheroid models were established. Data from previous studies report an increase in chemoresistance to therapeutic agents due to 3D spheroid growth compared to cancer cells grown as 2D monolayers ,50,70. CThe established neuroblastoma spheroid model—characterized by increased expression of a subgroup of CSC-like markers and increased Thr389 phosphorylation of the tumor-promoting RNA-binding protein La—was applied to assess the efficacy of the RIST treatment protocol, a novel multimodal treatment regimen for high-risk relapsed and refractory neuroblastoma, currently evaluated in a phase II clinical trial. Our results show the significant tumor-suppressive effect of the RIST treatment on cell viability and neoplastic signaling, emphasizing the importance of this efficient, reproducible, high-throughput 96-well 3D drug screening format to improve current treatment options for children with high-risk NB in the future.


Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine cutaneous malignancy that commonly affects older individuals with a high mortality rate . The treWhile the N6-methyladenosine (m6A) modification has not been documented in MCPyV mRNA, m6A residues are a known feature of the mRNAs of a closely related animal tumor virus, simian virus 40 (SV40), as well as numerous other viruses exhibiting nuclear replication . In eukaCancers, 2020, Orouji et al. addressed the regulatory role of YTHDF1 in the tumorigenesis of MCC, utilizing the genomic-wide analysis of seven MCC cell lines  [The dysregulation of the various components involved in m6A modification has been exhibited in several cancers, for instance, in the role of FTA in leukemia; however, no such regulatory mechanisms for m6A modification have been found for MCC as of yet . In theiegative) .Their experimental strategy included copy number analysis, which was performed using a panel of ~300 k genome-wide tag single-nucleotide polymorphisms (SNPs). Transcriptomic analysis was then performed using transcription data from different cell lines. Based on copy number and transcriptomic analyses, two genes on chromosome 20—YTHDF1 and KCNQ2—were identified and amplified. Since the expression of YTHDF1 was found to be more consistent among the tested cell lines than that of KCNQ2, YTHDF1 was studied further. To evaluate whether this finding was consistent at the level of protein expression, the levels of YTHDF1 proteins and its major paralog YTHDF2 were evaluated through immunoblots with lysates from MCC and melanoma cell lines. All MCC cell lines were found to exhibit higher expression of YTHDF1 and YTHDF2 proteins than the melanoma cell lines.The analysis was further focused on the immunohistochemistry of YTHDF1, which was performed on tissue microarrays from 31 MCC patients and 26 melanoma patients as the control cohort, revealing the YTHDF1 protein to be highly expressed in 74.2% of patient samples from MCC patients compared with only 3.8% of the samples from melanoma patients. To identify potential m6A sites in the clonally integrated sequence of the MCPyV genome of the WaGa cell line, a sequence-based RNA adenosine methylation site predictor (SRAMP) tool was used and a prediction score was determined. m6A modification was found to be present at two sites in the genomic sequence of MCPyV in the host cell. The role of YTHDF proteins was further explored through shRNA-mediated knockdown. Using two shRNAs, YTHDF1 and YTHDF2 were silenced in two MCC cell lines (WaGa and PeTa). Upon YTHDF1 depletion, the translational initiation factors eIF3A and eI3FB were found to be significantly downregulated. Finally, cell proliferation assays and colony formation assays were performed in MCC cell lines, revealing that the YTHDF1-silenced samples were less proliferative and showed less clonogenic potential compared with controls.To summarize, Orouji et al.  present This study is the first to elucidate the role of YTHDF1 in m6A identification in MCC and introduce a novel mechanism for the tumorigenesis of MCC involving the activation of cap-dependent translation. To further understand and validate the function of YTHDF1 in the tumorigenesis of MCC, future studies are needed to validate these findings. The study by Orouji et al.  notes th


Pyrazole derivatives, as a class of heterocyclic compounds, possess unique chemical structures that confer them with a broad spectrum of pharmacological activities. They have been extensively explored for designing potent and selective anticancer agents. In recent years, numerous pyrazole derivatives have been synthesized and evaluated for their anticancer potential against various cancer cell lines. Structure–activity relationship studies have shown that appropriate substitution on different positions of the pyrazole ring can significantly enhance anticancer efficacy and tumor selectivity. It is noteworthy that many pyrazole derivatives have demonstrated multiple mechanisms of anticancer action by interacting with various targets including tubulin, EGFR, CDK, BTK, and DNA. Therefore, this review summarizes the current understanding on the structural features of pyrazole derivatives and their structure-activity relationships with different targets, aiming to facilitate the development of potential pyrazole-based anticancer drugs. We focus on the latest research advances in anticancer activities of pyrazole compounds reported from 2018 to present. With rising living standards, the incidence of cancer has exhibited an upward trend, emerging as a leading threat to human health ,2. WhileNatural products play a pivotal role in anticancer drug research and development. Numerous naturally occurring heterocycles demonstrate unique antitumor activities, attributed to their structural rigidity imparted by distinct spatial conformations that mediate specific binding to cancer targets. Common heterocyclic scaffolds include pyrrole, furan, pyrazole, etc. . NotablyPyrazole is an important five-membered aromatic heterocyclic compound. Its unique chemical structure endows pyrazole with tremendous application value. The pyrazole ring consists of three adjacent carbon atoms and two nitrogen atoms, which impart polarity and reactivity to pyrazole due to the presence of nitrogen atoms. In addition, the pyrazole ring can undergo various substitution reactions. This provides ample opportunities for designing and developing novel anticancer drugs with pyrazole as the core scaffold.Pyrazole and its derivatives have demonstrated broad pharmacological activities, including anti-inflammatory, analgesic , antimicNumerous studies have shown that pyrazole derivatives possess good inhibitory activities against various targets in cancer cells, such as EGFR, VEGFR-2, CDK, BTK, and BRAF V600E, etc., which play an important role in anticancer drugs (details are provided below). In recent years, a number of pyrazole derivatives have been designed, synthesized, and evaluated for their anticancer efficacies in order to develop new anticancer candidates. Substituted pyrazoles, pyrazolines, pyrazolones, and pyrazolopyridines are several important classes of pyrazole derivatives. Among them, pyrazoline is a dihydro derivative of pyrazole, containing two adjacent nitrogen atoms and one endocyclic double bond in the ring. Pyrazolopyrimidine is one of the attractive fused heterocyclic pyrazole compounds. Pyrazolone is formed by attaching a ketone group at the 3-position of the pyrazole ring, retaining the coplanar structure of the pyrazole ring. The aforementioned three classes of compounds possess potent anticancer activity, as summarized in recently published reviews ,27,28. IMicrotubules are common hollow tubular fibrous structures in cells composed of tubulin, a small protein subunit . TubulinN-heterocyclic motif in celecoxib with the key structural features of CA-4 pyrazole derivatives to identify novel tubulin polymerization inhibitors pyrimidine analogs were reported by Li et al. as novel inhibition candidates of tubulin polymerization and screened for their antiproliferative activity on HeLa, MCF7, A549, HCT116, and B16F10 cancer cells with colchicine and paclitaxel as standard drugs pyrimidine derivatives based on the key pharmacophoric features of potent EGFR inhibitors pyrimidines and evaluated their antiproliferative activity against various human cancer cell lines including MCF7, A549, HeLa, and SiHa pyrimidine derivatives as promising anticancer agents warranting further investigation. And compound 20 with a 3,4,5-trimethoxyphenyl at C-7 and 5-F on the benzimidazole C-5 position was the most potent against MCF7, A549, HeLa, and SiHa with IC50 values of 3.2, 4.2, 8.9, and 7.9 µM, respectively. Immunostaining and Western blot analyses showed downregulation of EGFR, p-EGFR, STAT3, p-STAT3, and the anti-apoptotic protein Bcl-2 and procaspase-9, along with upregulation of pro-apoptotic proteins like p53, p21, and BAX, confirming these compounds could induce apoptosis. Compounds 18, 19, 20, and 21 also exhibited excellent EGFR inhibitor activity with IC50 values of 0.82, 0.31, 0.37, and 0.29 µM, compared to the positive control erlotinib (IC50 = 0.45 µM).Bagul et al. designed and synthesized a novel series of benzimidazole-linked pyrazolopyrimidine derivatives as potential EGFR inhibitors pyrimidine scaffold attenuated cytotoxicity, while incorporation of an aniline moiety at this position enhanced potency. Mechanistic studies showed that compound 24 induced apoptosis and cell cycle arrest at S and G2/M phases, it also markedly upregulated BAX gene expression and downregulated Bcl-2 expression. In summary, compound 24 displays promising antiproliferative and pro-apoptotic activities mediated through EGFR inhibition.Gaber et al. reported a series of novel 1hibitors . These c25 exhibited potent activity against all tested cancer cells with IC50 values ranging from 3.17 to 6.77 µM, superior to the reference drug axitinib. Structural–activity relationship analyses revealed that compounds with electron withdrawing groups on ring A or ring B showed the greatest growth inhibition. Notably, compound 25 displayed an IC50 value of 97 nM against VEGFR-2, comparable to the standard (IC50 = 39 nM). Mechanistically, compound 25 induces cell cycle arrest and apoptosis by depolarization of the mitochondrial membrane potential, increase in ROS, and damaging of DNA. Furthermore, it showed robust binding interactions with VEGFR-2 active sites and inhibited intersegmental blood vessel formation in transgenic zebrafish.VEGF and its receptor VEGFR-2 are critical regulators of endothelial cell proliferation and migration . A serie26 showed significant activity against all cancer cells tested, with IC50 values of 0.96, 1.40, and 2.16 µM for MCF7, A549, and DU145, respectively. This was superior to the standard drug etoposide. Compound 26 also showed low cytotoxicity against the normal breast cell line MCF10A. Structure–activity relationship studies indicated compound 26 exhibited more potent cytotoxicity with strong electron-donating methoxy substituents at the 3 and 5 positions of the phenyl ring. Additionally, compound 26 had higher inhibitory activity against VEGFR-2 tyrosine kinase (IC50 = 34.58 µM) compared to sorafenib. Docking study demonstrated that compound 26 showed more binding interactions with VEGFR-2 than VEGFR-1.Badithapuram et al. prepared a library of phthalazine-piperazine-pyrazole conjugates and tested their antiproliferative activity against MCF7, A549, and DU145 cells . Among t27 with an IC50 value of 16.50 µM compared to 23.31 µM for the standard drug tamoxifen. Some of these pyrazole derivatives reduced VEGFR-2 levels to varying degrees. Notably, compound 27 showed 78% inhibition (IC50 = 828.23 nM) of VEGFR-2, emerging as a promising anti-breast cancer drug candidate targeting this receptor. Further research demonstrated that compound 27 promoted pre-G1 apoptosis and inhibited G2/M phase growth by activating caspase-3, exhibiting significant pro-apoptotic activity.Dawood et al. prepared pyrazolone-pyrazole derivatives through a series of synthetic reactions and screened their in vitro inhibition on MCF7 . The bes28 showing superior potency compared to the positive control sorafenib. Compound 28 had IC50 values of 0.035, 2.24, and 0.028 µM in HCT116, UO31, and HepG2 cells, respectively, with no clear toxicity toward normal cells. Additionally, compound 28 (28–39) induced G2/M cell cycle arrest and overexpressed caspase-3, leading to apoptosis cell death in hepatocellular cancer cells. Analyses showed that compound 28 also possessed excellent CDK1 inhibitory activity with IC50 of 1.52 µM.Cyclin-dependent kinases (CDKs) are a family of serine-threonine kinases and play an important role in cell division, gene transcription, and other key biological processes . Harras a]pyrimidine compounds as potential CDK2 inhibitors pyrimidine derivatives and evaluated their antitumor efficacy toward HepG2, MCF7, and Hela pyridine derivatives and tested their antitumor activity pyrimidine derivatives as PIM-1 inhibitors and tested their antitumor activity toward HCT116 and MCF7 cells quinoline derivatives on haspin kinase across different cancer cell lines pyrimidine derivatives bearing carbon-aryl(heteryl)idene moieties as potential anticancer agents pyridine amide/amino acid functionalized derivatives and screened their in vitro antiproliferative activity against four cancer cell lines A549, MCF7, DU145, and HeLa pyrazole analogs as potential anticancer agents pyridine analogs and screened them for in vitro anticancer activities against HepG2, MCF7, and HeLa cancer cell lines pyrimidin-4(5H)-ones conjugated to a 1,2,3-triazole scaffold and evaluated their antitumor efficacy against C6 and U87 cell lines in vitro pyrazole derivative 87 and evaluated its in vitro anticancer activity against a panel of 17 human cancer cell lines pyrazine derivatives and tested for their in vitro anticancer activity against MCF7 cells pyrazole derivatives and in vitro cytotoxicity evaluation against MCF7 cells . All der93 exhibited good broad-spectrum inhibitory activity with IC50 values ranging from 1.8 to 4.4 µM, comparable to paclitaxel and camptothecin (IC50 < 0.8 µM).Verma et al. synthesized pyrazolacrylic oxadiazole and amide derivatives and evaluated their antiproliferative activity against HCT116, SW620, HT29, and MCF7 cell lines . Compoun94 displayed the highest cytotoxic activity (IC50 = 6.93 and 7.38 µM), comparable to doxorubicin (IC50 = 4.50 and 4.17 µM). The introduction of a cyclohexan-2,4-dien-1-one ring and ester group contributed to the remarkable anticancer activity of compound 94. Abdelgawad et al. synthesized novel substituted chalconated pyrazole analogs and tested their in vitro cytotoxicity against MCF7 and HepG2 cancer cells using MTT assay . Compoun95 exhibited promising inhibition of HepG2 (IC50 = 17.14 µM) and MCF7 (IC50 = 9.76 µM) compared to doxorubicin (IC50 = 4.50 and 4.17 µM). Replacement of the hydrogen atom at N-1 of the pyrazole with COCH3 or CHO drastically reduced the inhibitory activity.Ahmed et al. reported novel pyrazole, oxazole, and pyridine analogs containing naphthalene and furan motifs, and evaluated their antitumor activity against HepG2 and MCF7 cells . The pyr50 = 8.30 and 7.73 µM), compound 96 displayed superior inhibitory activity against HeLa (IC50 = 6.53 µM) and C6 cells (IC50 = 7.09 µM).Gezegen et al. synthesized a library of indenopyrazole derivatives and evaluated their inhibition of HeLa and C6 glioma cells . Compareb]pyridine hybrids and tested their in vitro cytotoxicity against HCT116 and MCF7 cells using MTT assay pyridine derivatives were prepared and evaluated for their antiproliferative activity against 60 human cancer cell lines by Naik et al. at the National Cancer Institute (NCI) pyridine scaffold represents a promising new class of anticancer agents with favorable drug-like properties.A novel series of pyrazol pyrazole derivatives against HCT116, HepG2, and MCF7 cells ,2,3triaz,4-d,2,3t108 (IC50 = 1.1 and 1.37 µM). This demonstrates that compound 108 may be a potential anticancer drug.Fathy et al. prepared new tetrahydroquinoline-pyrazole-hydrazide derivatives and assayed their in vitro anticancer activity against HepG2 and A549 cells . The hig109 exhibited potent activity against MCF7 breast cancer cells (GI = 49.88%) and moderate activity against T-47D cells (GI = 38.15%), while compound 110 displayed strong anticancer activity against UO-31 renal cancer cells (GI = 42.81%). Moreover, compound 111 showed strong inhibition of CCRF-CEM leukemia (GI = 41.37%) and SR leukemia cells (GI = 44.95%).Hassan et al. synthesized novel pyrazole derivatives and evaluated their in vitro anticancer activity against various human cancer cell lines using adriamycin as positive control . Compoun112, 113, and 114 (112–125) exhibited moderate to potent anticancer activities against certain cell lines. Compounds 112 and 113 showed higher COX-2 receptor affinity  than meclofenamine , conferring selectivity for COX.Hassan et al. synthesized fused pyrazolopyrimidines as pyrazoloquinazolines and benzoimidazo pyrazolopyrimidines, and tested them for antitumor activity on 60 human cancer cell lines . Among t115 and 116 exhibited remarkable potency against most of the tested cancer cell lines, with mean GI50 values of 5.47 and 2.24 µM, respectively. The broad-spectrum antitumor activities of compounds 115 and 116 against the NCI-60 panel highlight the pyrazolinylpyrazole and pyrazolinyltriazole scaffolds as promising new chemotypes for anticancer drug discovery.Ismail et al. reported a novel class of hybrids, including pyrazolinylpyrazoles and pyrazolinyltriazoles, and screened them for antitumor activity on the NCI-60 human tumor cell line panel . Compoun117 displayed the highest activity against HeLa, A549, MCF7, and IMR32 , in comparison with the positive control cisplatin .Kankala et al. synthesized a series of fluorophenylpyrazole-picolinamide analogs and evaluated their in vitro inhibitory activity against four human tumor cell lines: HeLa , A549 (lung), MCF7 (breast), and IMR32 (neuroblastoma) . Among t118, with IC50 values of 8.34 and 4.98 µg/mL, respectively, compared to the control drug doxorubicin. The enhanced cytotoxicity of compound 118 was attributed to its multiple active centers .Several new heterocycles containing chromone and pyrazole moieties were designed and prepared by Salem et al. . The in 119, 120, and 121 exhibited remarkable inhibitory activities against HeLa, PC3, and DU145 .Molnár et al. reported a novel series of A-ring-connected 2-pyrazoles of estradiol and evaluated their in vitro cytotoxicity against MCF7, PC3, DU145, and HeLa cancer cells . Compoun122 displayed significant cytotoxic activity against the tested cell lines with IC50 values ranging from 3.5 to 7.9 µM.Mótyán et al. synthesized novel D-ring-condensed 5-amino-1-arylpyrazoles and tested them for in vitro antiproliferative effects on human cancer cell lines including HeLa, U2Os, MCF7, PC3, and A549 . Among t123 exhibited the most potent anticancer activity against HepG2 with an IC50 value of 1.53 μM. Structure–activity relationship analyses revealed that the OH group of the curcumin/pyrazole curcumin scaffold was an important factor for the antitumor activity.Pham et al. prepared 20 curcuminoids and pyrazole-modified curcuminoids and evaluated their antiproliferation against the HepG2 cancer cell line . Compoun124 demonstrated good activity against the breast cancer cells T-47D.Kalavadiya et al. synthesized a class of 1,2,3-triazole containing pyrazolopyrimidine derivatives and screened them for in vitro anticancer activity against 60 cell lines . Compoun125 exhibited the most potent activity .Ravula et al. prepared novel isoxazole functionalized pyrazolopyridine derivatives and assayed them for their in vitro anticancer activity against cervical (HeLa), colon (COLO 205), liver (HepG2), and breast (MCF7) cancer cell lines . Among t126 (126–139) displayed that the best IC50 values were 6.9 and 13.6 µg/mL against both cell lines.Alsayari et al. synthesized new pyrazolothiazole-based heterocycles and evaluated their in vitro inhibition against HepG2 hepatocellular carcinoma and HCT116 colon carcinoma cell lines using the MTT assay . Compoun127 demonstrated the most potent antitumor activity with IC50 values of 19.38 and 10.70 µM against HeLa and SK-BR3, respectively. The hydrophobicity and binding ability of the core scaffolds were attributed to their antiproliferative effects.Chinthaparthi et al. synthesized tetrahydrodipyrazolo-pyridine derivatives and evaluated their in vitro antiproliferative effects on SK-BR3 and HeLa cancer cells . Compoun128 displayed promising inhibitory activity against A549 (IC50 = 1.90 µM) and HeLa (IC50 = 4.75 µM) compared to 5-fluorouracil . In addition, it showed lower activity against 293T (IC50 = 41.72 µM).Huang et al. designed and synthesized 3-phenyl-1-phenylsulfonyl pyrazoles containing an aminoguanidine moiety and screened them for their in vitro antitumor activity against A549 and HeLa cell lines . Compoun129 exhibited the most potent inhibition against both cell lines with IC50 values of 10.89 and 15.60 µM, respectively, compared to 5-FU (IC50 = 26.75 and 32.75 µM).Othman et al. synthesized pyrazolothiazole and thiazolopyridine analogs and evaluated their cytotoxicity against HepG2 hepatocellular carcinoma and MCF7 breast cancer cells . Compoun50 = 10, 9, and 11 µM, respectively) pyrimidines and evaluated their anticancer activity toward PC3, DU145, A549, and MCF7 cancer cell lines pyrazoles via Knoevenagel condensation and Michael addition and evaluated their in vitro anticancer activity against PC3 and SKOV3 cells using an MTT assay . All syn142 and 143 demonstrated remarkable antiproliferation against HeLa (IC50 = 3.60 and 4.61 µM), A549 (IC50 = 4.17 and 5.29 µM), and MDA-MB231 (IC50 = 3.94 and 4.92 µM), respectively.Several novel thiazolyl pyrazoles were synthesized and antitumor activity on HeLa, A549, and MDA-MB231 were evaluated by Mamidala et al. . Compoun144 exhibited good activity against 786-0 and MCF7 (IC50 = 9.9 and 31.87 µM), while compound 145 showed moderate activity against A431 and U-251 (IC50 = 19.98 and 25.78 µM).Parikh et al. synthesized and studied several new pyrano-pyrazole-5-carbonitriles and evaluated their cytotoxicity toward several carcinoma cell lines including 786-0 , A431 , MCF7 (breast), and U251 (glioblastoma) using a CCK-8 assay . Among t50 values against the tested cancer cells compared to the control drug etoposide. However, compound 146 exhibited significant cytotoxic activity against Panc1 with an IC50 value of 21.41 µM, lower than that of etoposide (IC50 = 24.35 µM).Sadeghian et al. prepared a library of pyranopyrazole derivatives and determined their anticancer activity against breast (MDA-MB231), colon (HT29) cancer cells, and normal human dermal fibroblasts (HDF) using an MTT assay . No comp147). Thus, compound 147 exhibited the highest inhibitory activity against MCF7 with an IC50 value of 145 µM/mL.Saleh et al. synthesized novel pyrazolopyranopyrimidine derivatives and assessed their antitumor properties against MCF7 breast cancer cells using an MTT assay . CompounH-pyrazole-4-carboxamide derivatives and evaluated their antitumor activity against A549, PC3, and K562 cancer cell lines [148 (IC50 = 10.2 µM) exhibited greater anticancer activity than the commercial anticancer drug 5-fluorouracil (IC50 = 23.1 μM) against A549. Meanwhile, compound 149 (IC50 = 12.4 µM) showed enhanced anticancer activity compared to Gefitinib (IC50 = 22.2 µM) against PC3.Xie et al. designed and prepared 25 new 5-trifluoromethyl-1ll lines . Among t150 demonstrated excellent cell growth inhibition even at different concentrations compared to doxorubicin.Xu et al. prepared and characterized some new fully substituted pyrazoles and evaluated their growth inhibitory activity against Huh7 cells . Among t151 exhibited greater cytotoxic activity than the final compounds against both cell lines, with increased sensitivity toward the A549 line.Otaibi et al. synthesized four novel hybrid compounds bearing pyrazole and chalcone moieties and evaluated their ability to inhibit the in vitro proliferation of human lung (A549) and colon (Caco2) cancer cell lines . Followi152 showed the highest 82% growth inhibition of the colon cancer cell line KM12 at 10 µM. Meanwhile, compound 153 inhibited the lung cancer cell line HOP92 with 43.19% growth inhibition.Asif et al. reported some novel spirooxindole-pyranopyrazole derivatives and evaluated their in vitro anticancer activity against 60 human cancer cell lines at the National Cancer Institute (NCI) . Compoun154 displayed significant potency against Hep2 and MCF7 with IC50 values of 8.85 and 10.80 µM, respectively, compared to the positive control 5-fluorouracil (IC50 = 7.19 and 10.19 µM).Azher et al. synthesized a library of substituted pyrazolopyrimidine analogs and tested their in vitro antitumor activity against several cell lines including MCF7, PC3, Hep2, and WI38 . Among t155 and 156 demonstrated good cytotoxic activity against MCF7 and HepG2 (IC50 = 3.89 and 8.42 µM). Additionally, compound 157 showed the highest inhibitory activity against HCT116 and HeLa (IC50 = 7.71 and 13.11 µM).Elmorsy et al. synthesized novel pyridopyrazolo-triazine and pyridopyrazolo-triazole derivatives and evaluated their cytotoxic activity against colon, hepatocellular, breast, and cervical carcinoma cell lines . Compoun158 and 159 displayed remarkable inhibitory activities toward all tested cancer cells with IC50 values ranging from 18.31 to 39.98 µM compared to the reference drug 5-fluorouracil.Hossan et al. prepared and characterized several novel functionalized pyrazolopyrimidine compounds and evaluated their in vitro antiproliferation activity against HepG2, Hep2, MCF7, and PC3 cancer cell lines and normal WI38 cells . CompounPyrazoles, as a class of nitrogen-containing heterocyclic compounds, possess abundant nitrogen atoms and aromatic rings in their core scaffolds, making them extremely promising lead structures for anticancer drug discovery. In recent years, various pyrazole derivatives have been extensively explored for anticancer drug development due to their unique chemical properties. Currently, pyrazole inhibitors targeting the epidermal growth factor receptor (EGFR) have attracted considerable attention. Future research on pyrazole derivatives as anticancer agents will likely focus on several key aspects: First, further optimization of the pyrazole core to improve binding affinity and selectivity against EGFR and other critical anticancer targets, such as HDAC, PI3K/AKT, and VEGFR; second, combination therapies with other anticancer agents to achieve synergistic therapeutic efficacy; third, development of antibody-drug conjugates utilizing the advantages of pyrazole small molecules to enhance tumor targeting; fourth, design of PROTACs and other novel targeted anticancer modalities; fifth, generation of new pyrazole compounds to overcome drug resistance mechanisms. In summary, pyrazole scaffolds hold tremendous potential for enabling impactful advances in anticancer drug discovery, and are poised to spearhead the development of a new generation of potent and safe anticancer therapeutics.Over the past few decades, drug discovery has become integral to medical research. The remarkable chemical diversity of heterocyclic compounds has attracted substantial scientific attention worldwide. Among them, nitrogen-containing heterocyclic pyrazole derivatives possess a broad spectrum of biological activities and therapeutic promise as prospective anticancer agents due to their unique chemical structures. In recent years, numerous pyrazole derivatives have been synthesized to target tumor-associated proteins including kinases, microtubule proteins, topoisomerases, and COX-2. Their anticancer activities were evaluated accordingly. Certain pyrazole compounds exhibited potent selective inhibition against targets, such as BRAF, EGFR, and other kinases. Additionally, pyrazole derivatives with varied structural skeletons demonstrated antiproliferative and pro-apoptotic effects across multiple cancer cell lines. Currently, some pyrazoles have advanced to clinical trials for treating several malignant cancers. However, most pyrazole anticancer candidates remain at the preclinical stage, necessitating further optimization to enhance efficacy and reduce toxicity. The pyrazole scaffold continues to be an important source for anticancer drug discovery. Other novel derivatives will likely find clinical applications in cancer treatment in coming years. Here, we comprehensively review and analyze over 150 individual pyrazole derivatives, providing a useful resource to stimulate more research into anticancer drug discovery using this versatile scaffold.