Klotho, a gene found on chromosome 13q12, is involved in a variety of processes and signaling pathways in the human body related to vitamin D metabolism; cardiovascular, renal, musculoskeletal, and skin diseases; and cancer biology. However, more importantly, it has been linked to beneficial effects related to anti-aging. The levels of soluble Klotho in the blood have been found to decline with age, increasing the risk of age-related diseases. When the Klotho gene was silenced or defective, it caused a shorter lifespan. However, when the gene was overexpressed, it resulted in a longer lifespan. Klotho has positive benefits on the neurological system by causing a higher representation of useful longevity genes, preventing further neuronal damage, and offering neuroprotection. Thus, it has the potential to become a new treatment for many age-related diseases that cause dementia, including multiple sclerosis, Alzheimer’s disease, and Parkinson’s disease. In this review, we discuss the mechanisms of Klotho’s benefits and roles on various organ systems, specifically on nervous system disorders that lead to dementia. Klotho is a membrane-bound anti-aging protein that functions as an endocrine, autocrine, and paracrine hormone targeting different cells . There aThe Klotho gene is greatly exhibited in the kidneys, where it modulates renal function, parathyroid glands, and choroid plexus ,4. It plDementia is characterized by global cognitive impairment, consisting of an acquired decline in cognitive and emotional functioning, as well as behavioral and psychiatric disturbances severe enough to interfere with daily life. It is one of the most prevalent clinical syndromes seen in the old age group (age >65 years) . It has In this article, we aim to further explore the associations between the Klotho gene and dementia and try to help build a platform so that work can be done to investigate the therapeutic potential of Klotho for neurodegenerative conditions such as Alzheimer’s disease.Role, synthesis, and functions of Klotho in humansKlotho, a gene present on chromosome 13q12, has three protein forms, namely, a secretory, a cell membrane-associated, and an intracellular form. Membrane protease can digest the extracellular Klotho domain and can be seen in CSF, urine, or blood . SecretoThe circulating levels of soluble Klotho have been observed to decrease with age, which increases the risk of age-related illnesses. Torbus-Paluszczak et al. 2018) demonstrated accelerated aging and a shortened lifespan in mice when the Klotho gene was silenced or deficient . In cont18 demonsExpression of Klotho in the nervous systemIn the CNS, a secreted version of Klotho plays an important role in neuronal activity. It is a critical component of processes such as aging and neurodegeneration. In CSF, the expression of cytokine/chemokine is suppressed by Klotho, thereby supporting the activation of neuronal cells.Klotho is abundant in hippocampus neurons, choroid plexus ependymal cells, Purkinje EC cells, and the white matter of the brain . Its relRole of Klotho on oxidative stress/free radical injury and age-related changesKlotho was identified as a product of an anti-aging gene . AccordiIn contrast, KL-OE is expected to prolong the lifespan by reducing oxidative stress. It works like a hormone; thus, it attaches to the membrane receptor and prevents insulin-like growth factor-1 (IGF-1) from sending an intracellular signal . Its conMechanism of action of Klotho and signaling pathwaysMany biological activities of Klotho have been found; however, their molecular processes are unknown and must be explained. Research has shown that circulating Klotho has a role in intracellular signaling cascades by modulating the activity of multiple cell surface receptors, including TGF-β1 ,19, p53/Klotho’s Effect on the TGF-β1 Signaling PathwaySeveral growth factors and cytokines induce epithelial-to-mesenchymal transition (EMT). TGF-1 is the most efficient and ubiquitous growth factor. It works by suppressing the expression of genes with epithelial activities such as E-cadherin and by increasing the production of genes with mesenchymal functions such as vimentin, collagen-1, and N-cadherin. TGF-1 signaling is disrupted by secreted Klotho, which binds directly to the type II TGF receptor and inhibits TGF-1 binding. Secreted Klotho inhibits EMT in cancer cells and prevents the spread of cancer ,31.Klotho’s Effect on the p53/p21 Signaling PathwayCell cycle arrest by tumor suppressor p53, which activates the downstream p53-dependent cyclin-dependent kinase factors p-16 and p-21, results in growth arrest and cell death. Klotho reduces cellular senility in MRC5 primary human fibroblast and human umbilical vascular endothelial cells by inhibiting the p53/p21-dependent pathway. According to one theory, Akt/protein kinase B (PKB) decreases cell lifetime via the p53/21-dependent pathway. However, these hypotheses need to be validated .Klotho’s Effect on the c-AMP/PKA Signaling Pathwayc-AMP is generated from ATP by the enzyme adenylate cyclase (AC). c-AMP is required for protein kinase A (PKA). It stays catalytically inactive throughout the typical cell cycle. When c-AMP levels rise, an active form of PKA is released, phosphorylating and regulating several target proteins, including enzymes. By promoting superoxide generation, angiotensin-II (ANG-II) contributes to endothelial dysfunction. Membrane Klotho increases the angiotensin-converting enzyme (ACE) activity by stimulating the c-AMP/PKA pathway in human umbilical vein endothelial cells. Overexpressed Klotho suppresses the nicotinamide adenine dinucleotide phosphate oxidase 2 expression, thereby reducing ANG-II-induced oxidative damage, superoxide production, and apoptosis in smooth muscle cells. Hence, Klotho protects smooth muscle cells .Klotho’s Effect on the PKC Signaling PathwayPKC is an isoenzyme that forms a family related to serine/threonine kinases that regulate the target proteins. 25-OH vitamin D3 1-α-hydroxylase gene expression is regulated by both c-AMP and PKC. Klotho, on the other hand, inhibits the gene for 25-OH vitamin D3 1-α-hydroxylase in kidney cells. It is believed that Klotho suppresses the 1-alpha hydroxylase genes through a c-AMP or PKC-independent pathway. However, Klotho activates the c-AMP and PKC signaling pathways. PKC signaling pathway activation only occurs in the kidney and testes. In contrast, c-AMP activation occurs in each cell line .Klotho’s Effect on the Wnt Signaling PathwayWnt1, a proto-oncogene, is an important secreted factor that is responsible for cell proliferation and its maintenance. Its continuous exposure leads to accelerated cellular senescence. The biological activity of Wnt is inhibited when it interacts with Klotho. This tumor suppressor effect of Klotho is due to the inhibition of Wnt signaling and p53/21 and insulin/IGF-1. Klotho plays a key role in tumor genesis, its progression, and its prognosis. In tumor cells, Klotho is silenced by histone deacetylation and by hypermethylation of its promoter. Low levels of Klotho are accompanied by raised levels of Wnt/beta-catenin, which lead to fibrogenesis. Consequently, the role of Klotho in CKD is evident, as it regulates Wnt/beta-catenin activity .Regulation of FGF-23 Signaling Via KlothoFGFR23 is a protein that targets the membrane expressions of Klotho and FGFR. FGF-23 initiates downstream signaling effects, such as GRB 2-associated binding protein 1 (GAB1), ERK1/2-SGK1, phospholipase C (PLC), Shc, fibroblast growth factor receptor substrate 2 (FRS2), or signal transducer and activator of transcription 1 (STAT1), through this complex, providing a likely mechanism for different genes to be expressed. According to some experiments, FGF-23 inhibits the biosynthesis of phosphate and vitamin D reabsorption in the proximal renal tubules. At the same time, its homozygous missense mutation in humans can lead to soft-tissue calcification due to severe hyperphosphatemia. Klotho and FGF-23, through apoptosis, prevent vitamin D-induced cell death and, through cell proliferation, prevent tissue atrophy. This signaling inhibits vitamin D-mediated cell death by downregulating 1-alpha hydroxylase gene expression and suppressing the caspase activity through phosphoinositide-3 kinase .Klotho’s Effects on the Insulin/IGF-1 Signaling PathwaysThe intracellular IGF-1 signaling cascade, which is blocked by the aging suppressor Klotho protein, extends lifespan. With Klotho binding, the insulin/IGF-1 receptor’s tyrosine kinase activity is blocked, followed by insulin receptor substrate inhibition (IRS). IRS inhibits PKB and phosphoinositol 3-kinase (PI3K) serine phosphorylation. The capacity of the Klotho protein to block the insulin/IGF1 receptor or the direct suppression of IRS, P13K, and PKB/Akt still needs to be determined. Klotho inhibits IGF-1-mediated signaling pathways, which suppress various tumor types. It stimulates the Foxo forkhead transcription factors, which are downregulated by insulin/IGF-1 signaling. Hence, it increases the mitochondrial MnSOD expression, eliminating the reactive oxygen species and providing oxidative stress tolerance in animals at the organismal and cellular levels. The Klotho hormone is also involved in oxidative stress resistance, which may add to its anti-aging properties .Relation of Klotho deficiency and neurological abnormalitiesWith age, both the expression and circulating levels of klotho decline. In a study by Xiao et al., Klotho levels decreased with age in human blood serum samples . Klotho Neuroprotective effect of Klotho in different types of dementiaDementia is a neurological disorder categorized by impairment in memory in addition to one or more cognitive domains . This impairment constitutes a decline from the previous level of function and should be significant enough to result in impairment in daily life .It is estimated that about 47 million people suffer from dementia worldwide. As age is the most important risk factor leading to dementia, this number is set to increase in the coming years . Other rAlzheimer’s disease is characterized by extensive cortical atrophy caused by the deposition of hyperphosphorylated tau protein and amyloid plaques. Interleukin-6 (IL-6) is thought to activate microglia, which, in turn, leads to an increase in tau phosphorylation and the production of acute-phase reactions in neural tissue. A positive correlation was found between Alzheimer’s disease and elevated IL-6 levels . Lewy-boAmyloid plaques in Alzheimer’s disease consist of β-amyloid which is created through the processing of APP. It is postulated that the extracellular processing of APP promotes Klotho expression which protects against the harmful effects of β-amyloid on the neurons. β-amyloid is also thought to damage neurons through reactive oxygen species, and Klotho has been illustrated to protect against these oxidative insults . Higher Klotho contributes crucially to protecting hippocampus neurons from amyloid formation and plays a role against glutamate toxicity. Klotho is also considered key for oligodendrocytes maturation and myelin integration. The role of Klotho in the cellular life span regulation by p53 repression and downregulating p21 protein levels may be related to enhancing signaling through the insulin/IGF-1 pathway is already known . On a miMultiple SclerosisMultiple sclerosis is a demyelinating condition causing inflammation and myelin sheaths destruction in different parts of the CNS. A study showed that serum Klotho concentration is higher in multiple sclerosis patients with a longer disease duration when compared to the control group . ZeldichAlzheimer’s DiseaseThe disease process is centrally associated with the accumulation of amyloid plaques, neurofibrillary tangles, and neurodegeneration of various neuronal pathways of the brain. Multiple theories such as hyperphosphorylated tau proteins abnormalities and oxidative stress initiate the disease progression. A study done in Baltimore demonstrated that there are lower Klotho levels in CSF of Alzheimer’s disease compared to the control group, lower levels in older adults compared to younger adults, and higher levels in males than females . PretreaCognitionAll types of dementia are neurodegenerative, and protein toxicity is the principal feature. The leading causes are Alzheimer’s disease and Parkinson’s disease causing deterioration of brain functions. Klotho expression is reported in the adult hippocampus, and it enhances cognitive performance in mammals . As mentParkinson’s DiseaseA study conducted in Iran showed findings denoting the neuroprotective role of exogenous Klotho in the rat model of Parkinson’s disease and the protective effect against astrogliosis, programmed cell death, and oxidative insults by different signaling cascades . ZimmermRole of Klotho in other organ systemsReduced serum Klotho level has been linked to a greater density of epicardial fat, carotid artery intima-media, and reduced flow-mediated dilation of the brachial artery in the cardiovascular system; hence, reduced levels of serum Klotho must be addressed as a preliminary prognosticator of atherosclerosis . The redIn the gastrointestinal system, the role of abnormal Klotho levels was observed in colorectal carcinoma. DNA hypermethylation specifically in the promoter region leads to suppression and notable downregulation of Klotho in malignant colon cells which leads to their multiplication. Adequate Klotho levels can cause apoptosis of malignant colon cells . Klotho In the renal system, Klotho deficiency is greatly linked with ionic imbalance, calcification in vessels, inflammation and fibrosis in the renal parenchyma, and skeletal disorders, which represents CKD. Stage 2 CKD shows a premature reduction in Klotho protein levels in the blood, and stage 1 CKD shows even an earlier premature reduction in urinary Klotho levels. Hence, deficient Klotho levels are responsible for the growth and succession of CKD and other complications. It shows an analytical capacity that acts as a demonstrative biomarker of renal disorders such as CKD . Any treKlotho is also found in the skeletal system, specifically in osteoblasts and osteoclasts. Klotho protein plays an important role as a co-receptor for FGF-23. It has an effective activity in the development of the skeletal system. Further research is required to use this discovery to form therapy for bone disorders such as osteogenesis imperfecta, osteoporosis, osteodystrophy related to renal system disorders, and other bone-related ailments .Therapeutic role of Klotho on dementiaA randomized control trial on middle-aged healthy males conducted in Slovenia (2019) showed that a lower dose therapy of fluvastatin and valsartan causes a higher representation of useful longevity genes sirtuin-silent mating type information regulation 2 homolog, protein kinase AMP-activated catalytic subunit alpha, and Klotho, so it can be expressed as a favorable novel therapeutic perspective for senile disorders . Dubal eKlotho is one of the essential proteins that function as neuroprotective, anti-apoptotic, and suppresses oxidative stress. Since the detection of this protein, multiple studies have expanded our understanding of the significant role Klotho plays in the aging process. The levels of Klotho level decrease with age, which, in turn, increases the risk of developing dementia. Maintaining optimum levels of serum Klotho can slow this process. Furthermore, Klotho has been found to reduce neural damage and neurodegeneration, thus protecting against cognitive decline and contributing to life extension in humans. However, further studies are needed to explore its therapeutic role in humans.


Introduction: The ligand-activated transcription factors, nuclear hormone receptors (NHRs), remain unexplored in hematological malignancies except for retinoic acid receptor alpha (RARA).Methods: Here we profiled the expression of various NHRs and their coregulators in Chronic myeloid leukemia (CML) cell lines and identified a significant differential expression pattern between inherently imatinib mesylate (IM)-sensitive and resistant cell lines.Results: Retinoid-X-receptor alpha (RXRA) was downregulated in CML cell lines inherently resistant to IM and in primary CML CD34+ cells. Pre-treatment with clinically relevant RXRA ligands improved sensitivity to IM in-vitro in both CML cell lines and primary CML cells. This combination effectively reduced the viability and colony-forming capacity of CML CD34+ cells in-vitro. In-vivo, this combination reduced leukemic burden and prolonged survival. Overexpression (OE) of RXRA inhibited proliferation and improved sensitivity to IM in-vitro. In-vivo, RXRA OE cells showed reduced engraftment of cells in the bone marrow, improved sensitivity to IM, and prolonged survival. Both RXRA OE and ligand treatment markedly reduced BCR::ABL1 downstream kinase activation, activating apoptotic cascades and improving sensitivity to IM. Importantly, RXRA OE also led to the disruption of the oxidative capacity of these cells.Conclusion: Combining IM with clinically available RXRA ligands could form an alternative treatment strategy in CML patients with suboptimal response to IM. Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by a reciprocal chromosomal translocation t that generates the BCR::ABL1 fusion protein, a constitutively active tyrosine kinase essential for the pathogenesis of the disease. Molecular targeted therapy with tyrosine kinase inhibitors (TKI) such as imatinib mesylate (IM) has dramatically improved the outcome of this disease. However, a proportion of patients fail to achieve or retain molecular response with TKIs leading to disease progression.The mechanism of TKI resistance in CML has been extensively studied and broadly classified as BCR::ABL1 dependent and independent. BCR::ABL1-dependent mechanisms include mutations in the BCR::ABL1 kinase domain and over expression of BCR::ABL1 or gene expression. BCR::ABL1 independent resistance mechansim include the persistance of leukemic stem cells, activation of multiple signaling pathways, and epigenetic events . MutatioRXRA) ligand bexarotene in combination with chemotherapy was more effective associated with better survival, and improved response in refractory cutaneous T-cell lymphoma (CTCL) (PPARG and RXRA were downregulated in chemoresistant acute myeloid leukemia (AML) cell lines  , metastaa (CTCL)  and non-a (CTCL) . The rolll lines , suggestRXRA, RXRB, and RXRG , vitamin-D-receptor (VDR), peroxisome proliferator-activated receptors (PPAR), liver-X-receptor (LXR), pregnane-X-receptor (PXR), and the constitutive androsterone receptor (CAR)  in SH-SY5Y cells  are members of the NHR superfamily . 9-cis rand RXRG . RXRs eior (CAR) . RARA ansyndrome . Severalll lines . AML pat, methyl thiazolyldiphenyl-tetrazolium bromide (MTT), 9-cis retinoic acid (9cRA), Bexarotene (Bexa), acitretin (ACI), SR11237, all trans retinoic acid (ATRA), testosterone, 17β-estradiol (17βE), pioglitazone (PIO), rosiglitazone (rosi), Triiodothyronine (T3) and GSK4716 were from Sigma-Aldrich . Rabbit monoclonal p-CRKL , p-STAT5 , p-AKT , caspase-3 , PARP , and RXRA  were from Cell Signaling Technologies . β-actin (A5441) was from Sigma-Aldrich , and horseradish peroxidase-conjugated goat anti-rabbit IgG , goat anti-mouse IgG  from Cell Signaling Technologies . Imatinib Mesylate used for mice model experiments is a gift from NATCO, India.2 at 37°C.CML cell lines JURL-MK1, KCL22, Lama84, KYO1, EM2, and KU812, were a kind gift from Dr. Markus Muschen, University of California San Diego (UCSD), with approval from DSMZ, Germany. Cell lines were cultured in RPMI medium with 10%-FBS and antibiotics (100 U/ml-penicillin and 100 μg/mL-streptomycin) in a humidified atmosphere with 5% CO+ cells, as reported previously . Primary CML cells from blood or bone marrow were processed and enriched for CML CD34eviously .Total RNA was extracted from CML cell lines  and treated with or without NHR ligands using Trizol reagent. RNA (1 μg) was converted to cDNA using random hexamer and reverse transcriptase enzyme .2 PCR array  (https://geneglobe.qiagen.com/product-groups/rt2-profiler-pcr-arrays). The expression of each NHR was normalized using five housekeeping genes , as recommended by the manufacturers. The data was analyzed using SA biosciences web-based analysis software .To identify the basal expression of NHRs and coregulators in CML cell lines, we used an NHR RTAHR (TaqMan assay ID: Hs00169233_m1), AR (Hs00171172_m1), ESR1 (Hs01046816_m1), ESRRG (Hs00976243_m1), PPARG (Hs01115513_m1), RXRA (Hs0107640_m1), RXRB (Hs 00232774_m1) and THRA (Hs00268470_m1) were evaluated by qRT-PCR. Imatinib influx transporter SLC22A1/hOCT1 (HS00427554-m1) expression was measured using TaqMan assay after treating the primary CML cells with NHR ligands. GAPDH was used as the housekeeping gene for these targets. Fold change in the expression of other NHRs such as RARA, PPARG, and VDR was measured by RQ-PCR using SYBR green chemistry (SYBR premix Ex TaqII (Tli RNAseH Plus), Takara, Bio, Shiga, Japan), and ACTB was used as the housekeeping gene to calculate the relative expression (∆∆Ct) of these targets.The expression of 5], were treated in a 96-well plate with increasing concentrations of imatinib (0–500 nM) for 48 h. Cell viability was measured at standard wavelength 570 nm and reference wavelength 630 nm using EL800 microplate reader with KC junior software .CML cell lines JURL-MK1, KCL22, Lama84, KYO1, EM2, and KU812 [1 × 10CML cell lines (KCL22 & Lama84) were pretreated with NHR agonists  of imatinib for 48 hrs.As suggested by previous reports , bulk pr+ cells (n = 39) and healthy donor CD34+ cells (n = 9) using total RNA purification plus kit . Expression of RXRA was assessed using nCounter, and data counts were normalized with housekeeping genes–ACTB, GAPDH & GUS using nSolver software .RNA was extracted from CML CD34+) or healthy donors  cells (2 × 105) were treated with and without RXRA ligands 9-cis retinoic acid, acitretin, or bexarotene in combination with imatinib for 24 h; 1 × 104 cells were seeded in methylcellulose medium . Colonies were visualized using a phase-contrast microscope (×10 objective) and enumerated after 14 days.Primary CML (bulk & CD34+ cells (2 × 105) were treated with RXR ligands 9-cis retinoic acid, acitretin, or bexarotene for 24 h, followed by imatinib for 48 h. Cells were washed with 1XPBS and stained with Annexin-V conjugated with allophycocyanin (APC) and 7-Aminoactinomycin D (7AAD) . BD-Accuri C6  with BD Accuri C6 software (Version 1.0.264.21) was used for flow cytometry analysis.Primary CML CD34CML cell lines  or primary CML cells were treated with RXRA ligands  for 24 h, followed by imatinib for 48 h. The whole-cell lysate was prepared using radioimmunoprecipitation assay (RIPA) buffer supplemented with protease-phosphatase inhibitor cocktail  and 2 mM phenyl methyl sulfonyl fluoride (Sigma Aldrich). Total protein concentration was measured using the Bradford assay. 50 ug of protein was resolved in a 10% SDS PAGE. Anti-p-CRKL, anti-p-AKT, anti-p-STAT5, anti-caspase3, and anti-PARP antibodies were used, and β-actin was used as the loading control.The RXRA plasmid containing the full-length cDNA, pLX304 (clone ID- HsCD00437134), was purchased from the DNASU plasmid repository . Lentivirus particles were prepared by transfecting 293T cells with the RXRA cDNA plasmid and packaging plasmids. Lentiviruses were collected after 48, 60, and 72 h, pooled together, filtered, and concentrated using a lenti-X concentrator , and stored at −80°C until use. Lama84 cells were transduced with lentiviruses in the presence of polybrene, followed by spinfection. RXRA OE virus transduced Lama84 cells were selected by blasticidin (1 mg/mL).6 cells) were then transplanted into sub-lethally irradiated (2.5Gy) 8–10 weeks old NSG mice  via tail-vein. Disease development was monitored by measuring luciferase expression by injecting D-luciferin substrate and imaging using an IVIS Spectrum in-vivo imaging system . One week after transplantation, the mice were treated orally with either imatinib mesylate (100 mg/kg dissolved in water)  or acitretin (10 mg/kg dissolved in corn oil) or in combination for 21 days. Engraftment of KCL22 cells in the mice was assessed by measuring luciferase expression every week for 6–7 weeks. All animal experiments were carried out per protocols approved by the Institutional Animal Care and Use Committee (IAEC No. 5/2019).293T cells were transfected with plasmid lentiX-Luc2 containing a luciferase reporter for lentivirus production. KCL22 cells were transduced with lentivirus containing the luciferase reporter. Luciferase transduced KCL22 (1*106) cells were transplanted into sub-lethally irradiated (2.5Gy) 8–10 weeks old NSG mice via tail-vein. The leukemic burden in peripheral blood was assessed using flow cytometry by measuring human CD45 expression . The percentage of survival of mice was evaluated by Kaplan-Meir survival analysis.RXRA/empty vector transduced Lama84 (2*105cells per well (4 replicates) were seeded in a 24-well XF24 plate coated with retronectin . Thirty minutes before analysis, the medium was replaced with Seahorse XF media , and the plate was incubated at 37°C. Three sequential measurements of OCR and ECAR were taken at basal, proton leak (oligomycin), maximal respiration (FCCP), and OXPHOS inhibition (rotenone and Antimycin-A) to assess bioenergetics.Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse-XFe24 Analyzer (Agilent Technologies). Briefly, Lama84 EV and RXRA OE 4 × 10t-test, non-parametric t-test, and ANOVA with Kruskal-Wallis’s correction were used where appropriate. All statistical analysis were done using GraphPad Prism-v5 software, and a p-value < 0.05 was considered statistically significant.Paired in-vitro cytotoxicity assay to identify CML cell lines that are sensitive or resistant to IM. KU812, EM2, and KYO1 cell lines were identified as sensitive, and JURL-MK-1, KCL22, and Lama84 cell lines as resistant to IM. The kill curves and IC-50 of the cell lines tested are listed  and ten coregulators  were upregulated and RORA & 4 coregulators  were downregulated in imatinib-sensitive cell lines. A Representative Heatmap illustrates the expression profile of NHRs and coregulators  were upregulated in at least one of the IM-sensitive cell lines  could improve IM sensitivity. The concentration of the ligands used for this treatment is listed . Treatmein-vitro in primary bulk cells obtained from imatinib naive CML patients  improved IM IC-50 patients . Other Npatients . Based oe status . Primarye status .+ cells in-vitro, and the cell viability and colony-forming capacity were assessed. RXRA ligands, either alone or combined with IM, significantly decreased cell viability , accelerated phase (AP), and blast crisis (BC) patient samples from the GSE4170 dataset and found significantly reduced expression of RXRA in BC-CML samples compared to CP and AP  in CML cell lines and primary cells. RXRA ligands decreased p-CRKL, p-AKT, and p-STAT5 at the protein level in combination with IM  in CML cnd generation TKIs, dasatinib and nilotinib  of RXRA in the Lama84 cells with deficient basal protein expression , showed a differential effect, sensitizing the KCL22 cell line but no impact on the Lama84 cell line in combination with IM. Similarly, THRA agonist T3 treatment sensitized Lama84 and did not affect KCl22. Although 17BE and stanozolol treatment improved viability in both cell lines, these ligands did not improve sensitivity to IM in bulk CML cells PPARG agonist pioglitazone. When we assessed the BCR-ABL downstream signaling pathways, we observed that RXRA agonist in combination with IM resulted in decreased p-CRKL, p-STAT5, and p-AKT along with activation of the apoptotic cascade in CML cell lines and primary cells.RXRA in imatinib-resistant Lama84 cell line showed decreased proliferation, decreased BCR-ABL downstream signaling, and improved imatinib sensitivity in-vitro. RXRA expression in CML CD34+ cells compared to HD CD34+ and a reduction in the expression of RXRA in blast crisis CML patients compared to chronic phase, accelerated phase CML from the GSE4170 dataset indicating a potential role of RXRA in hindering leukemogenic capacity in CML.Lentiviral-mediated overexpression of + cells have a high oxidative capacity, which could be inhibited using tigecycline , a more robust in-vivo model to evaluate the therapeutic efficacy of this combination and anti-leukemogenic potential RXRA has on CML LSCs is needed. We are currently exploring the mechanism by which RXRA activation by both molecular/pharmacological means could orchestrate the downstream kinase activities.While our findings suggest the potential use of RXRA agonists in combination with IM in eliminating CML CD34in-vitro and in-vivo results suggest that using RXRA ligands could be an effective combination therapy to improve the imatinib response in patients with CML.We demonstrated differential NHR expression between imatinib-resistant and sensitive cell lines. The ligand-dependent activation of RXRA could be an effective CML target, affecting BCR-ABL1 downstream signaling and apoptotic pathway. Our


DNAH5 and NME5, respectively, recapitulate the respective diseased phenotype on a molecular, structural and functional level.Primary ciliary dyskinesia (PCD) is a rare heterogenic genetic disorder associated with perturbed biogenesis or function of motile cilia. Motile cilia dysfunction results in diminished mucociliary clearance (MCC) of pathogens in the respiratory tract and chronic airway inflammation and infections successively causing progressive lung damage. Current approaches to treat PCD are symptomatic, only, indicating an urgent need for curative therapeutic options. Here, we developed an in vitro model for PCD based on human induced pluripotent stem cell (hiPSC)-derived airway epithelium in Air-Liquid-Interface cultures. Applying transmission electron microscopy, immunofluorescence staining, ciliary beat frequency, and mucociliary transport measurements, we could demonstrate that ciliated respiratory epithelia cells derived from two PCD patient-specific hiPSC lines carrying mutations in  DNAH5-mutated hiPSC-derived epithelial cells do not express the DNAH5 protein and consequently carry non-motile cilia with a reduced number of outer dynein arms, Sone et al. recently described the modeling of ciliary function using hiPSC-derived epithelial cells in more detail  = RTotal − RBlankR(2)2] = RTissue [Ω] × surface area [cm2]TEER [Ω × cmALI cultures were washed with PBS as described above and subsequently fixed and stored in 150 mM HEPES buffer containing 1.5% PFA and 1.5% glutaraldehyde. Membranes were cut off the ALI inserts and fixation and embedding were carried out as described in For the assessment of ultrastructural dynein arm defects, PCD Detect Software was used, following the developers’ workflow and using the same alignment settings . From eak (the fraction of the FOV containing MCC) of each FOV k using customized code in Matlab (Mathworks), as reported previously [k and Ck for all k = 1…n FOVs, respectively) per condition were compared with the non-parametric Kruskal–Wallis test, followed by Tukey-Kramer multi-comparison test, to test for different median values.Only cultures at day 35 of ALI or older were used for MCC measurements to ensure substantial maturity of MCC. To remove accumulated mucus and debris, ALI cultures were submerged with PBS for 10 min, then the PBS was carefully suctioned off. To visualize MCC, fluorescent microspheres  were diluted 1:1000 in PBS, and 20 µL of this suspension was added to the surface of each ALI culture. Particle movement was recorded using a Zeiss Axioscope fluorescence microscope equipped with an Orca Flash 4.0 camera  and a temperature-controlled chamber that was preheated to 37 °C. Apart from a few initial test recordings, standardized acquisition settings using a 10× objective were 1024 × 1024 pixels with 1.3 µm/pixel resolution at a framerate of 20 frames per second for a total duration of 10 s. For each condition, at least 3 experiments (different differentiations or clones (hiPSCs), or different isolations (pALI)) were performed. In each experiment, 5 fields of view (FOVs) with visible particle movement (sometimes only Brownian motion) were recorded from 2 insert cultures each. The particle trajectories were extracted from the raw movies using ImageJ/Fiji  with theeviously . For stamut and NME5mut cultures, as the movement of cilia could not be resolved at the tissue level. For tissue-level CBF measurements, ALI cultures were washed as above. High-speed movies of CBF (ca. 140 frames per second for a duration of at least 1.5 s) were recorded using a 40× long-distance phase contrast objective at a spatial resolution of 0.3 µm/pixel in a 512 × 512 pixel frame. For each condition, at least 3 experiments (different differentiations (hiPSCs) or different isolations (pALI)) were performed. From the cultures of each experiment, 6 FOVs with visible CBF were recorded. We measured cilia beat frequencies by applying Fourier spectral analysis, as previously described [Only cultures at day 35 of ALI or older were used for CBF measurements. CBF was recorded using a Zeiss Axioscope microscope, allowing for oblique phase contrast recordings, equipped with an Orca Flash 4.0 camera  and a temperature-controlled chamber that was preheated to 37 °C. Koehler illumination was set up for all recordings. Two different procedures were performed to measure CBF. In non-PCD pALI and non-PCD iALI cultures, CBF was measured at the tissue level, and single-cell CBF was measured in DNAH5escribed . The meaTM in a conical tube. DNase I at a concentration of 200 µg/mL was added to remove any free DNA resulting from the dissociation process. After 20 min warm culture medium was added at a ratio of 3:1 and the cells were centrifuged at 170 to 240× g for 5 min. The resulting pellet was resuspended in 1 mL culture medium to obtain a high cell density and 10 µL of this suspension was placed onto a glass bottom imaging dish . To confirm the viability of the cells, the same amount of life-dead stain  was added to the cell suspension droplet, and the solution was covered with a round glass coverslip in order to prevent evaporation. The measurements were performed using a 40× oil phase contrast objective (NA 1.4) under standardized acquisition settings, which were 800 frames per second for a duration of 2 s (NME5mut) and up to 10 s (DNAH5mut) of recording time and a spatial resolution of 0.16 µm/pixel in a 265 × 265 pixel frame. For each condition, at least 3 experiments (different differentiation (hiPSCs) or different isolations (pALI)) were performed, and in each experiment, at least 9 FOVs, i.e., 9 ciliated cells, were recorded. After each CBF movie recording a brightfield image and fluorescent images of the life-dead staining were recorded to confirm viability. The recordings were analyzed using the kymograph function in ImageJ. Kymographs of individual or groups of cilia were generated and the frequency was calculated by counting the amount of pixel intensity changes over time in the kymograph. Whenever a wave pattern was observed, the number of peaks were counted, else the dark lines were counted and divided by two to obtain a full beat cycle count. In the case of non-harmonic oscillation observed in DNAH5mut-derived ciliated cells, stationary vibrations were not included in the count. A median was calculated from all counted frequencies per ciliated cell. For statistical analysis, the pooled tissue-level medians and single-cell medians per condition were compared with the non-parametric Kruskal–Wallis test, followed by Tukey-Kramer multi-comparison test, to test for different median values.For single-cell CBF measurements, the cell layer was dissociated by incubating the ALI cultures for 20 min in prewarmed Accutasemut Clone 22 and Clone 24) and NME5 , respectively, were previously generated [For the establishment of a PCD in vitro disease model, two hiPSC lines from patients carrying homozygous mutations in DNAH5 , anterior foregut endoderm (AFE), and lung progenitor cells A. The prmut Cl. 22: 13.1 ± 4.1%; DNAH5mut Cl. 24: 14.0 ± 7.3% and NME5mut: 11.6 ± 3.1%) of the cells at day 14 of differentiation. In order to enrich lung progenitor cultures, magnetically activated cell sorting (MACS) using the cell surface maker carboxypeptidase M (CPM) was performed [mut Cl. 22: 50.2 ± 22.4%; DNAH5mut Cl. 24: 50.8 ± 14.7%; NME5mut: 57.4 ± 6.4%)  C.mut-derived cultures generated thicker cell layers and fewer AcTub, MUC5AC, and CCSP positive cells as compared to DNAH5mut and non-PCD diseased counterparts. Primary bronchial epithelial cell (PBEC)-derived ALI cultures served as control (non-PCD pALI). Additionally, airway epithelial cell-specific marker expression was confirmed by RT-qPCR  measurements were performed after 28 days of ALI culture and showed TEER values of 302–476 Ω × cm2, indicating intact and comparable barrier function in cultures derived from all cell lines , goblet cells (MUC5AC), club cells (CCSP) and basal cells (P63 and CK5)) in all cell lines D. Remark RT-qPCR E. Expresll lines F.In order to visualize ciliary protein expression, ALI cultures at day 28 post airlift were dissociated into single cells and stained for DNAH5 and NME5 protein, respectively. Immunofluorescence staining against AcTub was used to identify cilia and confirmed the presence of cilia with a normal appearance in all cell lines. In ciliated cells generated from non-PCD diseased primary cell-derived ALI cultures  of generated iALI cultures was performed. Analysis of cilia of non-PCD diseased hiPSC-derived cells showed a normal 9 + 2 ultrastructure pattern in more than 95% of analyzed cilia axonemes A,B as exFor investigating outer dynein arm (ODA) defects in DNAH5-mutated cilia, TEM cross-sections were analyzed using the PCD Detect software . This soTo evaluate the maturation and functional consequences of the DNAH5 and NME5 mutations in PCD, we measured ciliary beat frequencies (CBF) using high-speed microscopy on day 35 after airlift or later. In non-PCD cells (pALI and iALI) ciliary beat was visible directly in ALI cultures, enabling us to use tissue-level recordings and automated analysis of CBF A. In PCDSince functional MCC depends on many factors, including ciliary beat coordination and orientation ,40, CBF To date, mainly primary cells or non-airway immortalized cell lines  have been used for PCD in vitro modeling or drug screening ,47,48. HCurrent differentiation protocols for the generation of airway epithelium from hiPSCs often include an intermediate 3D culture step, in which lung progenitor cells are embedded in Matrigel droplets, prior to differentiation to airway epithelium ,26. HencDNAH11, HEATR2, and PIH1D3 using a complex airway-on-a-chip technology [Sone et al. previously reported a hiPSC-based disease model of PCD-causing mutations in chnology . By applchnology ,50, are chnology ,52. Morechnology  does notIn contrast, transwell ALI cultures are compatible with standard readouts, easier to establish due to the commercial availability and handling simplicity, and available in various formats including 96-well plates , allowing higher throughput screening of pharmacological compounds. Altogether, the airway-on-a-chip system is a valuable tool to investigate specific research questions under dynamic flow conditions; however, the transwell ALI culture system remains indispensable to be used as a complementary system, in particular for screening purposes.DNAH5 [NME5 [DNAH5 results in loss of DNAH5 protein, altered ODA ultrastructure, and reduced CBF, consistent with previously published iPSC-based models [DNAH5-mutated cultures. Moreover, we demonstrate the application of the newly developed PCD Detect software [DNAH5 can also result in less prominent ODA defects such as dynein arm truncation [DNAH5-mutated cultures. Altogether, the detailed characterization of our DNAH5-mutated iALI cultures serves as a proof-of-concept to demonstrate that our in vitro model recapitulates the mutDNAH5-specific phenotype and can be captured in detail by utilizing improved readout methods.In the present study, we describe the establishment of a hiPSC-based PCD in vitro disease model using patient lines containing mutations in the most frequently mutated and well-described PCD-associated gene DNAH5 ,42, and H5 [NME5 , respectd models . Additiosoftware  that allsoftware , but mutuncation . Hence, uncation  to identNME5, which was recently identified as PCD-associated. NME5 is part of the radial spoke neck; however, so far, only little is known about the function of NME5 and the pathomechanism of its mutation [NME5 in more detail. Our TEM results are consistent with the previously reported loss of central pair (9 + 0) in NME5-mutated cilia, and moreover, show additional ultrastructural abnormalities, such as outer doublet transposition  and presence of additional microtubules (9 + 4), which have not been described for this mutation before. Importantly, similar ultrastructural abnormalities were reported in other radial spoke protein defects [Next, we aimed at modeling the impact of mutations in the gene mutation ,53. Solemutation . Here, w defects ,54,55,56NME5 on single-cell and tissue levels, we performed high-speed video microscopy (HSVM). Single-cell functional analysis revealed that NME5-mutant ciliated cells beat in an uncoordinated way and exhibited partially circular cilia movements that resembled 9 + 0 nodal cilia beating. These nodal-like beat kinematics have been previously reported to be a consequence of central pair and transposition defects [NME5-mutated ciliated cells showed an increased variance of CBFs, compared to controls. By assessing the mucociliary transport capacity, we could additionally demonstrate that these alterations lead to a significantly diminished mucociliary clearance capacity (coverage and speed) compared to non-PCD controls (iALI and pALI). Altogether, these findings indicate that mutated NME5 not only impairs the beating synchrony and pattern of a given cell but also potentially prevents tissue-level metachronal wave formation due to divergent beat frequencies, which breaks down mucus clearance [To further investigate the effect of mutated  defects ,43,44,45learance .mutNME5 cells). Further underlying causes for the observed differences between pALI and iALI cultures may include an insufficient maturation of the iALIs. Despite analyzing the cultures earliest at day 35 after the airlift, maturation of ciliary beating  might be not fully completed. So far, the maturation of cilia beating during differentiation is not well understood, and investigation of bead transport over the course of differentiation and analysis of cilia length remains to be carried out for further understanding. In addition, the higher heterogeneity of hiPSC-derived cultures (ciliated cells appearing in clusters and/or uneven epithelium surface) compared to pALIs might reduce MCC coverage and average trajectory speed, since bead transport slows down in non-ciliated areas.Notably, also non-PCD iALIs showed reduced MCC compared to primary cell controls (non-PCD pALI), despite comparable CBF parameters (median and variance) and synchronous cilia beating. This heterogeneity may have been neglected in other studies that used smaller fields of view for measuring bead transport; e.g., Sone et al. used a 100 µm × 100 µm fields of view (FOV)  comparedTaking together, here we present a hiPSC-based PCD model platform utilizing a simple air-liquid interface culture system that recapitulates mutation-specific phenotypes by detailed characterization on a molecular  and functional  level. In addition, our system allows down-scaling of the culture format and up-scaling of cell yield, which are essential prerequisites for upcoming higher throughput screening applications.


ZDHHC1, a gene previously implicated in immunity and STING activation, as the top upregulated AA-specific gene under hypoxia. Together, these data reveal a differential chromatin landscape in TNBC associated with donor ancestry. The open chromatin structure of AA TNBC may contribute to a more lethal disease.African American (AA) women have an excessive risk of developing triple-negative breast cancer (TNBC). We employed Assay for Transposase-Accessible Chromatin using sequencing to characterize differences in chromatin accessibility between nine commonly used TNBC cell lines derived from patients of European and African ancestry. Principal component and chromosome mapping analyses of accessibility peaks with the most variance revealed separation of chromatin profiles by patient group. Motif enrichment and footprinting analyses of disparate open chromatin regions revealed differences in transcription factor activity, identifying 79 with ancestry-associated binding patterns (FDR < 0.01). AA TNBC cell lines exhibited increased accessibility for 62 transcription factors associated with epithelial-to-mesenchymal transition, cancer stemness/chemotherapeutic resistance, proliferation, and aberrant p53 regulation, as well as KAISO, which has been previously linked to aggressive tumor characteristics in AA patients with cancer. Differential Assay for Transposase-Accessible Chromatin signal analysis identified 1,596 genes located within promoters of differentially open chromatin regions in AA-derived TNBC, identifying DNA methyltransferase 1 as the top upregulated gene associated with African ancestry. Pathway analyses with these genes revealed enrichment in several pathways, including hypoxia. Culturing cells under hypoxia showed ancestry-specific stress responses that led to the identification of a core set of AA-associated transcription factors, which included members of the Kruppel-like factor and Sp subfamilies, as well as KAISO, and identified in vitro studies to advance health equity at all levels of scientific research.We identify an ancestry-associated open chromatin landscape and related transcription factors that may contribute to aggressive TNBC in AA women. Furthermore, this study advocates for the inclusion of diversely sourced cell lines in experimental  Under the proper growth conditions, cancer cell lines can retain many of the genetic properties of the parental tumor from which they were derived  patients  cell lines derived from women of two different ancestral backgrounds—EA and African American (AA). We chose to focus our investigation on TNBC due to the well-documented racial disparities in tumor biology and breast cancer survival . AA womeMycoplasma testing was not performed. The biological sex of all cell lines was female. To perform the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis, following regular cell culture procedure, one million cells  were suspended in 1.5 mL freezing medium . The above cryopreserved cells were sent to the Sequencing Facility at Frederick National Laboratory for Cancer Research, Frederick, MD, where sample processing and sequencing was done.All human breast cancer lines were obtained from ATCC. MDA-MB-157 , MDA-MB-231 , MDA-MB-436 , and HCC1806  cells were grown in DMEM (Gibco Laboratory) with 2 mmol/L glutamine (Millipore Sigma) and 10% FBS (HyClone Laboratories). MDA-MB-468 , Hs578T , HCC70 , BT549 , and HCC2157  cells were grown in RPMI media (Gibco Laboratory) supplemented with 10% FBS. HCC2157 cells grow in suspension whereas all other cell lines are adherent. We obtained authentication of these cell lines through ATCC services using a short tandem repeat analysis; as cell lines were obtained directly from ATCC, 2) conditions, cells  were cultured at 37°C in a 5% CO2 humidified environment as an adherent monolayer. Hypoxia experiments were performed with the above eight adherent cell lines in a hypoxia chamber (BioSpherix) at 1% O2 at 37°C in a 5% CO2-humidified environment. Briefly, breast cancer cells were plated in T75 flask (1 × 106 cells/flask). Media was changed after 24 hours, and cells were exposed to normoxia (21% O2) and hypoxia (1% O2) for 48 hours. Experiments were conducted in duplicates. After 48 hours, the cells were harvested from the flasks by 0.25% trypsin (Gibco Laboratory) and 1 million cells were suspended in 1.5 mL of freezing medium . The above cryopreserved cells were sent to the Sequencing Facility at Frederick National Laboratory for Cancer Research, Frederick, MD, where sample processing and sequencing were done.For normoxic (21% O2 in water). Cells were centrifuged at 500 rcf for 5 minutes in a prechilled (4°C) fixed-angle centrifuge. After centrifugation, 900 µL of supernatant was aspirated, which left 100 µL of supernatant. This remaining 100 µL of supernatant was carefully aspirated by pipetting with a P200 pipette tip to avoid the cell pellet. Cell pellets were then resuspended in 50 µL of ATAC-seq RSB containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin by pipetting up and down three times. This cell lysis reaction was incubated on ice for 3 minutes. After lysis, 1 mL of ATAC-seq RSB containing 0.1% Tween-20 (without NP40 or digitonin) was added, and the tubes were inverted to mix. Nuclei were then centrifuged for 10 minutes at 500 rcf in a prechilled (4°C) fixed-angle centrifuge. Supernatant was removed with two pipetting steps, as described before, and nuclei were resuspended in 50 µL of transposition mix by pipetting up and down six times. Transposition reactions were incubated at 37°C for 30 minutes in a thermomixer with shaking at 1,000 rpm. Reactions were cleaned up with Zymo DNA Clean and Concentrator 5 columns. The remainder of the ATAC-seq library preparation was performed as described previously . Sequencing was performed on an Illumina NextSeq 2000 machine at the Sequencing Facility, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research using standard protocols. Briefly, 16 mRNA sequencing samples were pooled and sequenced on NextSeq 2000 P2 using Illumina Stranded mRNA Ligation Kit and paired-end sequencing. The samples had 56 to 69 million pass filter reads with more than 95% of bases above the quality score of Q30. The RNA sequencing (RNA-seq) data for the human breast cancer cell lines were deposited in the NCBI's GEO database under accession number GSE223181.After verifying that the pair-end reads are of acceptable sequencing quality, sequencing adapters and primers were trimmed away using Cutadapt . TrimmedThe dimensionality of the Tn5-nicking site counts matrix was reduced by calculating the principal components and plotting the first two components which explain a significant proportion of the overall variation.Loading the count matrix into DESeq2 v.1.45.5 , were then imported into the Enrichr tool  to identify groups of TF proteins that have known and/or predicted relationships . Using Sq value (FDR) < 0.05, the absolute value of fold change > 2. We then performed gene set enrichment analysis (GSEA) using the gene transcription regulation reference (Gene Transcription Regulation Database: GTRD) from the Molecular Signatures Database: http://www.gsea-msigdb.org/gsea/msigdb/human/collections.jsp).The Illumina FASTQ files were assessed for quality control using FastQC v.0.11.6. Reads of the samples were trimmed for adapters and low-quality bases using Cutadapt before alignment with the reference genome (hg38) and the annotated transcripts using STAR v.2.7.5b. The gene expression quantification was performed using RSEM v1.3.2. The average mapping rate of all samples was 97% and unique alignment was above 88%. The mapping statistics were calculated using Picard software. Percent coding bases were between 51% and 61%. Percent untranslated region bases were 32%–43%, and mRNA bases were between 89% and 94% for all the samples. Library complexity was measured in terms of unique fragments in the mapped reads using Picard's MarkDuplicate utility. The samples had 58%–71% nonduplicate reads. Differential expression analysis was done using DESeq2 package in R. Differential expression genes (DEG) were filtered by a https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/Software.cgi), which is a fast distance–based method to infer ancestry based on references from multiple genotype datasets, including those of populations of European, African, and Asian descent. The method assigned a predominant African ancestry to MDA-MB-468, MDA-MB-157, HCC1806, and HCC70 cells and a predominant European ancestry to the MDA-MB-231, MDA-MB-436, Hs578T, and BT549 cells, consistent with donor ethnicities reported by ATCC for these cell lines. Ancestry estimates of our cell lines aligned closely with publicly available data from the Estimated Cell Line Ancestry (ECLA) database from the Moffitt Cancer Center (http://ecla.moffitt.org).Germline variants were called using GATK's HaplotypeCaller v.4.2.6.1 from available RNA-seq data for eight cell lines cultured under normoxic conditions, MDA-MB-468, MDA-MB-157, HCC1806, HCC70, MDA-MB-231, MDA-MB-436, Hs578T, and BT549. For admixture analysis, only SNPs that were biallelic were retained in the analysis. We used GRAF-pop v.1.0 (https://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/BRCA/20160128/) covering 112 TNBC patient tumors  to infer differences in TF activity by genetic ancestry, which was assigned using previously reported quantified genetic ancestry estimates  breast cancer data were downloaded from the Broad Institute's GDAC  and AA (n = 5) donors of nine commonly used TNBC cell lines (http://ecla.moffitt.org) indicates an average European ancestry of 93% for the four EA TNBC cell lines and 79.5% West African ancestry for the five AA TNBC cell lines (2FC| > 2) in EA compared with AA TNBC cells confirmed distinctive patterning of open chromatin across a number of chromosomes  and BCL6B (Pnom = 0.26). In AA, 11 TFs overlapped, including SNAI1 (Pnom = 0.19), NR1H4 (Pnom < 0.0001), MSX2 (Pnom = 0.03), and KLF14 (Pnom = 0.08). Taken together, these data suggest that TF activities show significant differences by donor ancestry, with African ancestry displaying an ancestry-specific TF activity profile associated with increased aggressiveness in breast cancer.Next, we performed a digital footprinting analysis of differentially open chromatin regions using TOBIAS. We uncovered significant differences in TF binding by ancestry  and AA (n = 34) patients within TCGA to perform an upstream regulator analysis to infer critical transcriptional regulators based on ancestry-related differences in downstream gene expression , JUNB , BATF , and FOSL1 . In TNBC tumors from AA patients, validated TFs totaled 25 and notably included TP53 , TP63 , TCF3  and TCF4 , TFAP2A , and various members of the FOX TF family. These patient data were concordant with some of the top TFs identified in our cell line data.We next sought to extend these findings from human TNBC cell lines to TNBC patient tumors to investigate if ancestry-specific TF activity showed similar patterning by patient ancestry group. ATAC-seq data were not available within TCGA for validation of TF binding and motif enrichment analyses; however, we leveraged RNA-seq data from triple-negative breast tumor tissues in EA , a long noncoding RNA suspected to function as a tumor suppressor, followed by LRIG1, which interestingly also has an anti-invasive role in basal-like breast cancer cells through encouraging mesenchymal-to-epithelial transition , a DNA methyltransferase  upregulated in promoter regions. In EA TNBC, enriched Hallmark-annotated cancer pathways (FDR < 0.2) included epithelial-to-mesenchymal transition (Padjusted = 2.3 × 10−4), downregulation of UV response (Padjusted = 0.04), complement (Padjusted = 0.1), and hedgehog signaling  are negative regulators of this process , apical junction (Padjusted = 2.9 × 10−3), and hypoxia (Padjusted = 0.1), among others  and hypoxic (1% O2) conditions and performed ATAC-seq. Genome-wide analysis of differentially accessible chromatin sites (FDR < 0.01 and |log2FC| > 2) in EA compared with AA TNBC under hypoxia revealed distinctive patterning of open chromatin across a number of chromosomes , with a high degree of TF coexpression and shared functions  among the small network ; as expected, DETFs from AP-1 family, namely the JUN and FOS TF subfamilies, showed highly robust interactions, likely reflecting the homodimeric and heterodimeric functional relationships between these proteins. We performed a pathway enrichment analysis using REACTOME based on the TF network analysis to identify shared biologic function of significant TF clusters (P < 1.0 × 10−16), which was more expansive and extensive than that of the EA PPI network . TP53 is one of the most prominent nodes featured in this network, with high degree of connectivity and central to several other key nodes, such as SP-1, EGR-1, and KLF-4. In pathway enrichment analyses using REACTOME, 33 pathways were identified with an FDR < 0.01, 15 of which are shown in To better understand how differentially bound TFs involved in the ancestry-driven hypoxia response might act in concert to dictate downstream gene expression, we constructed a protein-protein interaction (PPI) network using the STRING database , allowinclusters ; the topMAGI1, a junctional scaffold protein that acts as a tumor suppressor in the context breast cancer through inhibition of the p38 stress pathway . For 111/112 TCGA samples used in our study, genetic ancestry consensus was concordant with SRR. Genetic ancestry consensus classification of African or admixed African ancestry captured 100% of TCGA participants who self-identified as “Black or African American”. Genetic ancestry consensus classification of EUR ancestry captured 98.7% of TCGA participants who self-identified as “White”; in one case, a participant that self-identified as White demonstrated a higher proportion of admixed American genetic ancestry. Among TCGA participants who self-identified as Black or African American, the average percentage of African ancestry was 83.2%, which is comparable to the quantified genetic ancestry estimates for the human cell lines used in this study that were classified as African American-derived (average African ancestry of 79.5%). Among TCGA participants who self-identified as White, the average percentage of European ancestry was 96.7%, which is comparable to the quantified genetic ancestry estimates for the human cell lines used in this study that were classified as European American-derived (average European ancestry of 93.0%).Click here for additional data file.Supplementary Figure 2Supplemental Figure 2. Expression of genes by donor ancestry in TNBC cell lines. Top-ranked genes identified to be located in open chromatin regions with an ancestry relationship were individually confirmed to be differentially expressed at the transcript level in matched RNA-seq data from the same human TNBC cell lines. Significance testing with two-sided t-test.Click here for additional data file.Supplementary Table 1Supplementary table 1 displaying data output from motif enrichment analysis performed on ATAC-seq data from TNBC cell lines.Click here for additional data file.Supplementary Table 2Supplementary table 2 displaying data output from digital footprinting analysis performed on ATAC-seq data from TNBC cell lines.Click here for additional data file.Supplementary Table 3Supplementary table 3 displaying data output from gene set enrichment analysis for transcriptional regulators performed on RNA-seq data from TNBC cell lines.Click here for additional data file.Supplementary Table 4Supplementary table 4 displaying data output from upstream regulator analysis performed on RNA-seq data from TNBC patients in TCGA.Click here for additional data file.Supplementary Table 5Supplementary table 5 displaying data output from differential ATAC signal performed on ATAC-seq data from TNBC cell lines.Click here for additional data file.Supplementary Table 6Supplementary table 6 displaying data output from differential gene expression analysis performed on RNA-seq data from TNBC cell lines.Click here for additional data file.Supplementary Table 7Supplementary table 7 displaying data output from digital footprinting analysis performed on ATAC-seq data from TNBC cell lines cultured under hypoxia.Click here for additional data file.Supplementary Table 8Supplementary table 8 displaying data output from differential ATAC signal analysis performed on ATAC-seq data from TNBC cell lines cultured under hypoxia.Click here for additional data file.


To ensure health equity, it is imperative that cell lines representing different lung cancer histological types, carrying different cancer driver genes, and representing different genders, races, and ethnicities should be available. This is particularly relevant for cell lines from Black men, who experience the highest lung cancer mortality in the United States. Here, we undertook a review of the available lung cancer cell lines and their racial and ethnic origin. We noted a marked imbalance in the availability of cell lines from different races and ethnicities. Cell lines from Black patients were strongly underrepresented, and we identified no cell lines from Hispanic/Latin(x) (H/L), American Indian/American Native (AI/AN), or Native Hawaiian or other Pacific Islander (NHOPI) patients. The majority of cell lines were derived from White and Asian patients. Also missing are cell lines representing the cells-of-origin of the major lung cancer histological types, which can be used to model lung cancer development and to study the effects of environmental exposures on lung tissues. To our knowledge, the few available immortalized alveolar epithelial cell lines are all derived from White subjects, and the race and ethnicity of a handful of cell lines derived from bronchial epithelial cells are unknown. The lack of an appropriately diverse collection of lung cancer cell lines and lung cancer cell-of-origin lines severely limits racially and ethnically inclusive lung cancer research. It impedes the ability to develop inclusive models, screen comprehensively for effective compounds, pre-clinically test new drugs, and optimize precision medicine. It thereby hinders the development of therapies that can increase the survival of minority and underserved patients. The noted lack of cell lines from underrepresented groups should constitute a call to action to establish additional cell lines and ensure adequate representation of all population groups in this critical pre-clinical research resource.Lung cancer is the leading cause of cancer death in the United States and worldwide, and a major source of cancer health disparities. Lung cancer cell lines provide key  Lung cancer remains the leading cause of cancer death in the United States , 2 and iin vitro or in vivo , squamous cell carcinoma (LUSQ), small cell lung cancer (SCLC), and large cell carcinoma (LULCC) –42, the https://seer.cancer.gov/) program, a large United States-based cancer registry that at present includes over 331 million subjects from 17 regions, to assess lung cancer mortality for different races/ethnicities for the main histological types . Current recommendations are to test cell lines for mycoplasma and authenticity when they first reach a new laboratory, before publication, and every two months while in culture.Two important considerations when using cell lines for research are cell line quality and authenticity. The presence of contaminating microorganisms, particularly mycoplasma, and the cross-contamination with other cell lines can invalidate performed research , 50. MycWe investigated the availability of lung cancer cell lines representing different races/ethnicities using the resources listed in We identified 390 cell lines from Asian lung cancer patients , H/LWe found almost 300 cell lines for which race/ethnicity is unknown –73.We noted that the number of cell lines developed from men was over 2-fold higher than cell lines developed from women, and this excess was most prominent for the cell lines developed from Asian individuals   were established using SV40LgT antigen  and small airway epithelial cells (SAECs)  , 86. BEAThe availability of methods to establish immortalized alveolar and airway cells allows progress to be made in deriving additional cell lines from racially and ethnically diverse subjects. However, there is one important cell type for which no immortalized human cell lines have yet been established: pulmonary neuroendocrine (PNE) cells, the main cell-of-origin of SCLC . ImmortaDerivation of cell line types from induced pluripotent cells has also been used to obtain bronchial epithelial cells  and alvein vitro cultures can be useful for the study of cancer driver and tumor suppressor genes, epigenetic changes in cancer cells, the effects of environmental exposures, and the investigation of lung cancer risk SNPs, among other topics. Cell lines provide a relatively pure population of cells compared to heterogeneous tumor or tissue samples that can contain variable amounts of contaminating blood cells and stroma. This simplification can greatly facilitate analyses and provides one powerful strategy to leverage cell-based models. However, it lacks the complexity arising from growth in three-dimensional space or from the interactions with other cell types, such as fibroblasts and blood vessels. Growth of pure cell lines with a defined medium in three dimensions can provide the next level of complexity, while the addition of fibroblasts, endothelial, and blood cells can further simulate in vivo characteristics. Even further advanced are three-dimensional models, so-called “organs-on-a-chip”, which may incorporate an air-liquid interface and/or the movement associated with breathing  mice or humanized mice need to be used. Such models, known as xenografts, can be made using human cancer cell lines, primary patient tumors, or even circulating tumor cells , the number of sequenced lung cancer samples from White patients outnumbers that of Black patients by almost 9:1. Thus, data on driver mutations in underrepresented patients must also be expanded. Clearly, much work remains to be done. The first step is to highlight current shortcomings in knowledge and resources, and to disseminate information to lung cancer patients of all races and ethnicities about the need for cell lines representing lung cancer in their communities. Explaining how lung cancer cell lines and cell-of-origin lines can be used to improve research and develop new therapies for people of the patients’ own racial/ethnic backgrounds can help patients make an informed decision about whether to participate. In addition, it would be beneficial if the donations of tissues/cells were discussed with patients by researchers and/or clinicians from their own racial/ethnic group, supporting mutual trust and a better understanding of research goals  , Black non-Hispanic, White non-Hispanic (IAO), and Asian .


To investigate the mechanism of exosomes' role in the transformation of MDS to AML.Exosomes in culture supernatants of MDS and AML cell lines, were extracted by ultrafiltration and identified in three ways: morphology, size, and exosome protein surface markers. Exosomes from AML cell lines were then co-cultured with MDS cell lines and their impacts on MDS cell microenvironment, proliferation, differentiation, cell cycle, and apoptosis were analyzed by CCK-8 assay and flow cytometry. Furthermore, exosomes from MSC were extracted for further authentication.The transmission electron microscopy, nanoparticle tracking analysis, Western blotting, and flow cytometry methods all verify that ultrafiltration is a reliable method to extract exosomes in the culture medium. Exosomes from AML cell lines inhibit the proliferation of MDS cell lines, block cell cycle progression, and promote apoptosis and cell differentiation. It also leads to increased secretion of tumor necrosis factor-α (TNF-α) and reactive oxygen species (ROS) in MDS cell lines. In addition, MSC-derived exosomes were found to inhibit the proliferation of MDS cell lines, arrest cell cycle progression, promote apoptosis, and inhibit differentiation.Ultrafiltration is a proper methodology in extracting exosomes. The exosomes of AML origin and MSC origin may play a role in MDS leukemia transformation via targeting TNF-α/ROS-Caspase3 pathway. Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders that are characterized by inefficient hematopoiesis, hemocytopenia, and an increase in blast cells, with nearly one-third of patients at risk of progression into acute myeloid leukemia(AML) , 2. In tExosomes are extracellular vesicles 30–150 nm in diameter formed by cells by way of endocytosis . ExosomeApoptosis is a programmed cell death caused by the death receptor pathway or the mitochondrial pathway . Tumor nAt present, there is still no effective therapeutic methodology in treating MDS. The low and intermediate risk MDS is mainly managed via hypomethylating agents and supportive care, such as transfusion therapy. Cytotoxic chemotherapy, targeted therapy, and immunomodulatory therapy are the common managements for high-risk MDS, Allogeneic hematopoietic stem cell transplantation (HSPC) is the only treatment possible to cure MDS, but the treatment-related mortality risk and the source of stem cells are still the biggest obstacles , 23.It is now generally accepted that tumor cell-derived exosomes can promote disease progression and metastasis . In this2.The human myelodysplastic syndrome cell lines SKM-1 and MUTZ-1 were purchased from Otwo Biotech , and the human myeloid leukemia cell lines THP-1, K562, and HL-60 are routinely maintained in our laboratory. SKM-1 was cultured in IMDM with 10% FBS and 1% penicillin, MUTZ-1, THP-1, K562 and HL-60 were cultured in RPMI-1640 with 10% FBS and 1% penicillin, and all cell lines were cultured at 37 °C and 5% COWhen density reached 60–70%, cells were collected by centrifugation at 300 g, 4 °C, 10 min, the supernatant was collected after 48 h of further incubation in a serum-free medium. The supernatant was centrifuged at 300 g, 4 °C, 10 min to remove cellular components; 2000 g, 4 °C, 10 min to remove dead cells; then 10000 g,4 °C,30 min to remove cell fragments. The supernatant was filtrated via a 0.22 μm filter to remove apoptotic vesicles. The exosomes were collected by ultrafiltration using a 10 KD ultrafiltration tube . Finally, the ultrafiltrate was resuspended in PBS to elute the medium components.Transmission electron microscope (TEM) and Nanoparticle Tracking Analysis (NTA) were sent to Beijing Ovison Gene Technology Company for analysis. Exosome proteins were lysed using RIPA lysate . Rough quantification was performed using a BCA protein concentration assay kit . Equal amounts of exosome protein (60 μg) were subjected to polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferring the protein onto 0.45 μm PVDF membranes . Using 5% skimmed milk powder for closure, incubated with primary antibody CD63, TSG101, Calnexin for 1.5 h and HRP-labelled goat anti-rabbit secondary antibody for 2 h at room temperature in a shaker. The results were finally developed using the ECL  chemiluminescence method. Exosomes were identified by flow cytometry method using CD9 -FITC, CD63- PE, CD81- FITC, CD33- APC, and CD14- APC antibodies as the surface markers.PKH26  binds to the lipid region of the cell membrane, showing as a sign of red fluorescence. The labeled exosomes were co-cultured with the MDS cell line for 48 h and then observed under a fluorescent microscope and photographed for documentation.5 cells were taken in a flow tube, 5 μl FITC, and 5 μl PI were added, mixed, and incubated for 15 min at room temperature and protected from light and then tested on the machine. Cell differentiation and proliferation indicators were detected using CD11b-PE and CD117- PC5 antibodies. The Seven-item Cytokine Assay Kit  was used to detect cytokines in the culture supernatant. Detection of intracellular reactive oxygen levels were used the Reactive Oxygen Species Assay Kit , Briefly, the cells were resuspended in DCFH-D diluted with serum-free medium, incubated in a 37 °C cell incubator for 20 min, washed with serum-free medium and then assayed on the machine.AML cell line-derived exosomes were co-cultured with MDS cell lines for 48 h before flow cytometric analyses of cell cycle, apoptosis, differentiation, cytokines, and ROS. Cells were fixed in pre-cooled 70% ethanol at 4 °C, stained using propidium iodide (PI) according to the Cell Cycle Assay Kit , and DNA content was determined by flow cytometry analysis, and the percentage of cells in G0/G1, S, and G2/M phases were counted for comparison. Apoptosis staining was performed using the Apoptosis Assay Kit . 103/well with 100 μ cell suspension per well, incubate at 37 °C, 5% CO2. The plate was removed at 0 d, 1 d, 2 d, 3 d, and 4 d, and 10 μl of CCK-8 reagent was added to each well and the culture was continued for 2 h. The absorbance (OD) values of the corresponding wells were measured at 450 nm and the cell proliferation curves were plotted.Cell proliferation was measured using the Cell Counting Kit (CCK-8) . AML cell line-derived exosomes were co-cultured with the MDS cell line for 48 h and then inoculated into 96-well plates at 5 × 10Proteins from MDS cell lines were extracted using RIPA lysate  after 48 h co-culturing The extracted proteins were quantified using the BCA protein concentration assay kit . Equal amounts of protein (60 μg) were subjected to SDS-PAGE and subsequently transferred onto 0.45 μm PVDF membranes . The primary antibody Caspase3 and β-actin were incubated for 1.5 h on a shaker at room temperature. Then the HRP-labelled secondary antibody was incubated for 2 h. The final development was performed by chemiluminescence using ECL .t-test. p < 0.05 was considered statistically significant.SPSS (version 17.0) and GraphPad (Prism 7.0) were used for statistical processing and plotting. All data were obtained from three independent experiments and the means of the two samples were compared using the Exosomes as described above were scanned by transmission electron microscopy, the exosomes were membrane vesicles less than 200 nm in diameter and resembling a teacup tray-like shape Fig. A, B. NanTo identify whether exosomes of AML cell line could be translocated into the MDS cell line, the exosomes labeled with PKH26 were cultured with the MDS cell line. The result showed that the MDS cell line can take up the PKH26-labelled exosomes after two days of incubation  and that K562-exo made SKM-1 cells to stop in the G0/G1 phase (p < 0.001)  and K562-exo resulted in MUTZ-1 cells being blocked in the S phase (p < 0.001)  and K562-exo (p < 0.001) co-culturing increased SKM-1 cell apoptosis   Fig. A. In conp < 0.05, p < 0.001)  and MUTZ-1 cells (P < 0.01) and suppressed the expression of the proliferation marker CD117 (P < 0.001) in SKM-1 cells compared to the control, which was consistent with the results of the CCK-8 assay. Similarly, CD117, a cell proliferation marker, was not detected in MUTZ-1 cells  after 48 h of co-culture  , while the rest of the cytokines did not differ significantly or even showed a decreasing trend  Fig. A, which One-third of MDS patients will transform into AML, and 24% of MDS patients have been reported to die as a result of leukemic transformation , HoweverWe first extracted AML cell line-derived exosomes by ultrafiltration and then characterized them by transmission electron microscopy, nanoparticle tracking analysis, protein blotting, and flow cytometry. Next, AML cell line-derived exosomes were co-cultured with MDS cell lines, and exosomes were also labeled with PKH26 to demonstrate that the exosomes could be taken up by MDS cell lines and function as intercellular communication. After co-culturing for 48 h and detection of cell proliferation by CCK-8, we found that exosomes of AML cell line origin inhibited the growth of MDS cell lines, which we further verified with CD117, a protein closely related to proliferation, also known as c-kit , THP-1-eTo further investigate how AML cell line-derived exosomes cause apoptosis in MDS cell lines, we again examined the alteration of seven cytokines and reactive oxygen species after 48 h co-culturing. We found that co-culturing resulted in more secretion of TNF-α and ROS from MDS cell lines. TNF-α is a key ligand in the death receptor signal pathway and ROS is an important signaling molecule in the mitochondrial pathway, both of which activate the Caspase protease family in a cascade reaction that ultimately activates Caspase3 protein to cause apoptosis . We alsoMesenchymal stem cells (MSC) are multipotent stem cells with the ability of self-renewal and multi-directional differentiation, which are widely distributed in many tissues of the human body, such as bone marrow, umbilical cord, and fat . Some stIn conclusion, our study shows that exosomes derived from AML cell lines can inhibit the proliferation of MDS cell lines, block cell cycle progression, promote apoptosis and differentiation, and lead to increased secretion of TNF-α and ROS in MDS cells. The data from this study suggests that AML-derived exosomes can affect the apoptosis of MDS cell lines via the TNF-α/ROS-Caspase3 pathway, thus affecting the transformation of MDS to AML Fig. . We also


Altered MM cytogenetics did not affect dinaciclib response ex vivo, alone or in combined treatment, suggesting that these combinations could be a suitable therapeutic option for patients bearing cytogenetic alterations and poor prognosis. This work also opens the possibility to explore cyclin‐dependent kinase 9 inhibition as a targeted therapy in MM patients overexpressing or with high dependence on MCL‐1.A better understanding of multiple myeloma (MM) biology has led to the development of novel therapies. However, MM is still an incurable disease and new pharmacological strategies are needed. Dinaciclib, a multiple cyclin‐dependent kinase (CDK) inhibitor, which inhibits CDK1, 2, 5 and 9, displays significant antimyeloma activity as found in phase II clinical trials. In this study, we have explored the mechanism of dinaciclib‐induced death and evaluated its enhancement by different BH3 mimetics in MM cell lines as well as in plasma cells from MM patients. Our results indicate a synergistic effect of dinaciclib‐based combinations with B‐cell lymphoma 2 or B‐cell lymphoma extra‐large inhibitors, especially in MM cell lines with partial dependence on myeloid cell leukemia sequence 1 (MCL‐1). Simultaneous treatment with dinaciclib and BH3 mimetics ABT‐199 or A‐1155463 additionally showed a synergistic effect in plasma cells from MM patients,  L notably reduce cell resistance to death.Multiple myeloma (MM) remains an incurable disease. As a general rule, MM cells overexpress or are highly dependent on MCL‐1 to survive. Therefore, dinaciclib (a multiple CDK inhibitor) decreases MM cells viability due to CDK9 inhibition and subsequent MCL‐1 downregulation. Rational combinations with corresponding BH3 mimetics in cases of codependence on BCL‐2 or BCL‐X MM is characterized by the appearance of multiple malignant plasma cell foci in the bone marrow (BM) causing bone destruction, thrombocytopenia and neutropenia . VirtualCyclin‐dependent kinases (CDKs) are serine/threonine kinases that regulate progression through cell cycle, by means of complexes with specific cell cycle regulatory proteins called cyclins . Aside f50) in the low nanomolar range . These proteins sequester proapoptotic members of the same family inhibiting mitochondrial outer membrane permeabilization (MOMP)  for 30 min on ice. Solubilized proteins were resolved by SDS–15% PAGE and transferred to nitrocellulose membranes. Afterward, membranes were blocked in TBS‐T  containing 5% skimmed milk and incubated with primary antibodies  diluted in TBS‐T containing 5% bovine serum albumin and 0.05% NaN3. Primary antibodies anti‐MCL‐1 (sc‐12756 and sc‐819) and anti‐BAK (sc‐832) were provided by Santa Cruz Biotechnology, Inc. . Primary anti‐BAX antibody (#2772) and anti‐BCL‐XL  were supplied by Cell Signaling Technology . Finally, membranes were washed with TBS‐T and incubated with the corresponding peroxidase‐labeled secondary antibodies (Sigma‐Aldrich) in TBS‐T 2.5% skimmed milk and revealed by Amersham™ Imager 600 . Control of protein loading was achieved by reprobing with anti‐α‐tubulin mAb . Band quantification was performed using imagej software .Changes in the amount of cytosolic or mitochondrial proteins were determined by western blot of cell extracts. After treatment with the corresponding drugs (3 × 102.5−/− and Bak−/−) were selected by culture in media containing puromycin (2–3 μg·mL−1) followed by limiting dilution. As a control, MM.1S cells were infected and puromycin selected with lentiviral particles containing empty LentiCRISPR v2 vector (MM.1S Ø). Accordingly, BAX−/−, BAK−/− and DKO were confirmed by western blot.MM.1S cell line was serially spin infected  with le2.64 cells (U266 and MM.1S) were treated in a flat bottom, 96‐well (200 μL per well) plate with dinaciclib in combination with BH3 mimetics  in complete medium mixed with GrowDex™ matrix 0.25%. After 24 h of incubation, supernatant was discarded and 20 μL per well of GrowDase™ , mixed with complete RPMI 1640 medium, was added. GrowDase™ was then incubated for 3 h at 37 °C. Finally, samples were filtered through Corning® cell strainers (40 μm pore size), cells stained with annexin V‐FITC/PI and analyzed by flow cytometry.U266 and MM.1S were cultured in 3D system by using GrowDex™  as a 3D matrix. For that purpose, 6 × 102.7m pH 7.4, NaCl 140 mm, CaCl2 2.5 mm and 0.5 μL of annexin V‐FITC per condition. Recombinant annexin V was purified and conjugated with FITC in our laboratory following Logue et al. procedure . In some cases, the Chou‐Talalay method [compusyn software, supplied by ComboSyn, Inc., Paramus, NJ, USA) to determine possible synergies. Additionally, receiver operating characteristics (ROC) test was used for validating cut‐off values between patients' samples cohorts.Statistical analysis was performed by using  authors , 40, 41,y method  was also33.1m) for 24 h. Analysis of cell proliferation showed that U266 and MM.1S cells were more resistant to dinaciclib than NCI‐H929, OPM‐2 and RPMI 8226   Table . Dinacic0) Table . After 40) Table .Mitochondria have a key role in cell death and survival. BAX and BAK are the main proapoptotic proteins of the BCL‐2 family, which turn from harmless monomers into deadly oligomers causing MOMP . As repoL , S63845 (a MCL‐1 inhibitor) or A‐1155463 (a BCL‐XL inhibitor). We adjusted single drug concentration for each line so that the amount of cell death was not greater than 30%. The difference between OBS and EXP specific apoptosis for each combination was calculated as indicated in Section L and indirect inhibition of MCL‐1 could overcome double combination resistance in this cell line.We next tried to improve the We also analyzed the effect of dinaciclib combinations with BH3 mimetics in 3D culture models generated with the cell lines more resistant to dinaciclib (U266 and MM.1S). Dinaciclib combinations with BH3 mimetics were quite efficient in promoting cell death in these 3D models, as illustrated in Fig. 3.3m palbociclib and 72 h incubation were needed to induce death in MM.1S and NCI‐H929 cells. Furthermore, palbociclib‐treated cells seemed to acquire a senescent‐like phenotype with bigger size and internal microvesiculation . As illustrated in Fig. + cells with 12 nm dinaciclib. This concentration was close to the LD50 of the majority of the MM cell lines tested (Table m) dinaciclib concentration than samples from SMM patients; however, the differences among them were not statistically significant. At a higher concentration (12 nm), samples from MGUS patients were significantly less sensitive than those of SMM, MM or PCL  and Din‐S those whose death rates were upper than the first quartile. Both cut‐off values were validated by ROC analysis  and dinaciclib‐resistant (Din‐R) according to their is Table . As illuex vivo sensitivity to dinaciclib. No significant differences were found between Din‐S and Din‐R subgroups regarding the previous lines of treatment , t, t, t, t, gain/amplification 1q (+1q), TP53 alterations and IGH‐FGFR3/IGH‐MAF gene fusions. In this regard, no significant differences were found between both groups, suggesting that sensitivity to dinaciclib is independent of the presence of these recurrent MM cytogenetic alterations ; second, samples showing moderate apoptotic rates with each single drug that were potentiated by drug combinations, and, finally, insensitive cells to combinations of dinaciclib with BH3 mimetics . The difference between OBS and EXP specific apoptosis was just calculated for samples classified in the second group. As shown in Fig. ex vivo, significantly increased when combined with ABT‐199 or A‐1155463. We could observe clear ex vivo synergy in 13/36 (36.1%) samples for ABT‐199 or A‐1155463 dinaciclib‐based combinations, and in 6/35 (17.1%) samples for S63845 combinations.Since combination of dinaciclib and BH3 mimetics showed synergism in MM cell lines, we next analyzed whether this was also the case in plasma cells from myeloma patients. As illustrated in Fig. ex vivo could correlate with the response to combinations. As in the previous analysis, we only considered samples that were not sensitive to single drugs but displayed a positive synergy to combinations between dinaciclib and BH3 mimetics. As illustrated in Fig. We would also like to analyze whether the resistance or sensitivity to dinaciclib treatment As previously mentioned, MM samples were classified into two groups attending to their cytogenetic status. Again, for this analysis, we only considered samples that did not show high sensitivity to single drugs. In this sense, no statistically significant differences between patients with FISH negative or altered cytogenetics were found in the response to combinations of dinaciclib with BH3 mimetics Fig. . Interes4Throughout the last decades, a great improvement of the myeloma therapeutic toolkit has occurred. The introduction of targeted drugs, such as proteasome inhibitors and cell therapies resulted in an increase of the median overall survival . HoweverL and increases apoptosis rates in some MM cell lines and plasma cells from MM patients, ex vivo. This has been previously found with other CDK inhibitors and BH3 mimetics. In particular, Zhou et al. [L in soft‐tissue sarcomas. The combination of dinaciclib with ABT‐199 has also been proposed against acute lymphocytic leukemia [Interestingly, we observed that dinaciclib synergizes with BH3 mimetics binding to BCL‐2 or BCL‐Xu et al.  found syu et al.  found a leukemia .ex vivo inhibition of MCL‐1 [L was involved in observed synergy [It is well‐known that CDK9 plays a main role in the control of basal gene transcription, thus assuring cell transcriptional homeostasis . Previouof MCL‐1  and MCL‐of MCL‐1 . When coof MCL‐1 , 27, 44. synergy . The maiMoreover, Din‐R group of plasma cells from MM patients exhibited low sensitivity to S63845, according to the proposed link between dependence on MCL‐1 and dinaciclib sensitivity. Additionally, partial dependence on BCL‐2 could also be related to the sensitivity to dinaciclib, as we found in our samples from patients. Within this context, we also observed that KMS‐12‐BM cell line, considered to be highly dependent on BCL‐2 , was sende novo patients and 70% of relapsed/refractory patients [m than those of SMM, MM or PCL. Our work is the first to evaluate the sensitivity to MCL‐1 indirect inhibitor depending on disease stage of MM and so it is not possible to compare with previous works. In particular, it has been reported that in MGUS the prevalence of +1q is lower than in SMM or MM patients, as determined by FISH and next‐generation sequencing techniques [Multiple myeloma genome instability not only facilitates clonal proliferation and spread of disease, but also creates cell vulnerabilities which can be used to develop new therapeutic strategies. In this study, we have evaluated if cytogenetic alterations were associated to dinaciclib response in MM cells. In this sense, we could not observe any significant correlation between genetic aberrations and response in MM cells, either in cell lines or in plasma cells from patients. However, plasma cells displaying +1q were highly sensitive to dinaciclib, alone or in combination with BH3 mimetics. 1q21 amplification is one of the most common cytogenetic alterations occurring in around 40% of patients . The prepatients , and it chniques , 72, 73.5ex vivo analysis of particular characteristics of malignant plasma cells. In this sense, dynamic BH3 profiling (DBP) has emerged as a useful tool to identify cellular dependence on individual antiapoptotic proteins with a simple FACS analysis or a microfluidic‐based DBP device [L respectively. In this regard, reversible thrombocytopenia caused by A‐1155463 [L inhibitors [The future of MM treatment relies on precision medicine, with decision making based on the P device , 75. As P device . This obP device . Combina‐1155463  may be phibitors . To achihibitors , 78, 79.hibitors  which wiThe authors declare no conflict of interest.MB‐V, JN and IM designed the study; MB‐V, NJ‐A, MC, AS‐DV and AB performed sample analysis; MB‐V, VP, AA, JN and IM interpreted and analyzed data; RD, GA and LP collected samples, reviewed patient records and collected data; MB‐V and IM wrote the manuscript. All authors had full access to all data, carefully reviewed the manuscript and approved the final version.https://www.webofscience.com/api/gateway/wos/peer‐review/10.1002/1878‐0261.13522.The peer review history for this article is available at Fig. S1. Validation and functional characterization of engineered MM cell lines.Click here for additional data file.Fig. S2. Analysis of cell death induced by combinations of dinaciclib and BH3 mimetics in MM cell lines.Click here for additional data file.Fig. S3. OBS and EXP specific apoptosis induced by dinaciclib‐based combinations with BH3 mimetics in MM cell lines.Click here for additional data file.Fig. S4. Synergy validation between dinaciclib and BH3 mimetics in U266 and MM.1S cell lines by CI calculation through the Chou‐Talalay formula.Click here for additional data file.Fig. S5. OBS–EXP specific apoptosis for triple combinations of dinaciclib, ABT‐199 and A‐1155463 in OPM‐2 and RPMI 8226 cells.Click here for additional data file.Fig. S6. Cell death induced by dinaciclib and BH3 mimetics combinations in MM cell lines partially dependent on MCL‐1 in 3D culture models.Click here for additional data file.Fig. S7. Cell death induced by combinations of NVP‐2 (CDK9 inhibitor) and BH3 mimetics in MM cell lines.Click here for additional data file.Fig. S8. Effect of CDK4/6 inhibition on MM cell lines.Click here for additional data file.Fig. S9. Cell death induced by dinaciclib‐based combinations with BH3 mimetics in Din‐S and Din‐R subgroups of patients' samples.Click here for additional data file.Table S1.In vitro dinaciclib lethal dose (LD50) in established multiple myeloma (MM) cell lines used in this study.Click here for additional data file.Table S2. Validation of dinaciclib‐resistant (Din‐R) and dinaciclib‐sensitive (Din‐S) cut‐off values: ROC analysis.Click here for additional data file.Table S3. Summary of samples with high‐risk cytogenetic alterations used in this work. Dinaciclib resistance (Din‐R) or sensitivity (Din‐S) of each sample is also indicated.Click here for additional data file.