Depletion Of c-Myc Research Articles

SARS-CoV-2 Accessory Protein Orf7b Induces Lung Injury via c-Myc Mediated Apoptosis and Ferroptosis.

The pandemic of coronavirus disease 2019 (COVID-19) has been the foremost modern global public health challenge. The airway is the primary target in severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) infection, with substantial cell death and lung injury being signature hallmarks of exposure. The viral factors that contribute to cell death and lung injury remain incompletely understood. Thus, this study investigated the role of open reading frame 7b (Orf7b), an accessory protein of the virus, in causing lung injury. In screening viral proteins, we identified Orf7b as one of the major viral factors that mediates lung epithelial cell death. Overexpression of Orf7b leads to apoptosis and ferroptosis in lung epithelial cells, and inhibitors of apoptosis and ferroptosis ablate Orf7b-induced cell death. Orf7b upregulates the transcription regulator, c-Myc, which is integral in the activation of lung cell death pathways. Depletion of c-Myc alleviates both apoptotic and ferroptotic cell deaths and lung injury in mouse models. Our study suggests a major role of Orf7b in the cell death and lung injury attributable to COVID-19 exposure, supporting it as a potential therapeutic target.

The 5-/12-lipoxygenase-BLT2 cascade induces elevated expression of c-Myc, thus mediating the proliferation and migration of KRAS mutant colorectal cancer cells

ABSTRACTColorectal cancer (CRC) is the most common malignant tumor and one of the leading causes of cancer-related death worldwide. Oncogene KRAS is a commonly mutated in CRC and plays crucial roles in the colorectal tumorigenesis. Emerging evidence suggests that increased expression levels of c-Myc are critical for KRAS-mediated CRC progression. However, it is unclear exactly how c-Myc expression is regulated in CRC. In the present study, we found that blockade of 5/12-lipoxygenase (5/12-LO) or leukotriene B4 receptor (BLT2) markedly reduced c-Myc expression in KRAS-mutant LOVO cells, while the control COLO 320DM cells were not affected. When the 5/12-LO products LTB4 and 12(S)-HETE, ligands of BLT2, were added to LOVO cells, c-Myc expression levels were restored, together suggesting that ‘5/12-LO-BLT2 cascade’ regulates c-Myc expression in KRAS-mutant CRC cells. We also observed that ‘5/12-LO-BLT2’-mediated c-Myc upregulation contributes to cell proliferation by regulating the expression of cyclin D1 in LOVO cells. Moreover, ‘5/12-LO-BLT2-cMyc’ cascade regulates the expression of EMT-related proteins in KRAS-mutant CRC cells, and depletion of both c-Myc and BLT2 using siRNA significantly enhanced the level of E-cadherin and reduced the level of Vimentin in LOVO cells. Taken together, our findings suggest that the BLT2-linked cascade promotes KRAS-mutant CRC cell proliferation and migration through the elevated expression of c-Myc. Thus, our results suggest that BLT2 appears to be an effective therapeutic target for KRAS-mutant CRCs.

Dimethyl alpha-ketoglutarate inhibits proliferation in diffuse intrinsic pontine glioma by reprogramming epigenetic and transcriptional networks

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor with limited therapeutic options. Here, we investigated the potential of dimethyl alpha-ketoglutarate (DMKG) as an anti-proliferative agent against DIPG and unraveled its underlying molecular mechanisms. DMKG exhibited robust inhibition of DIPG cell proliferation, colony formation, and neurosphere growth. Transcriptomic analysis revealed substantial alterations in gene expression, with upregulated genes enriched in hypoxia-related pathways and downregulated genes associated with cell division and the mitotic cell cycle. Notably, DMKG induced G1/S phase cell cycle arrest and downregulated histone H3 lysine 27 acetylation (H3K27ac) without affecting H3 methylation levels. The inhibition of AKT and ERK signaling pathways by DMKG coincided with decreased expression of the CBP/p300 coactivator. Importantly, we identified the c-MYC-p300/ATF1-p300 axis as a key mediator of DMKG's effects, demonstrating reduced binding to target gene promoters and decreased H3K27ac levels. Depletion of c-MYC or ATF1 effectively inhibited DIPG cell growth. These findings highlight the potent anti-proliferative properties of DMKG, its impact on epigenetic modifications, and the involvement of the c-MYC-p300/ATF1-p300 axis in DIPG, shedding light on potential therapeutic strategies for this devastating disease.

Abstract 4900: Preclinical efficacy of targeting epigenetic mechanisms in AML with 3q26 lesions and EVI1 overexpression

Abstract EVI1 gene is located at 3q26.2 in the MECOM locus and encodes for a zinc-finger transcription factor. Chromosome translocation t(3;3) or inv(3) at 3q26 repositions the enhancer (E) of the tumor suppressor GATA2 to induce EVI1 over-expression, while concomitantly repressing GATA2. EVI1 over-expression promotes self-renewal and blocks differentiation of leukemia stem-progenitor cells (LSCs) and causes therapy refractoriness and inferior overall survival in AML. Previous reports highlighted that targeting BRD4 with BET inhibitor (BETi) is active against AML cells with inv(3)/t(3;3). BETi treatment also represses EVI1 and its targets c-Myc and Bcl-xL. We recently reported that tegavivint (TV) (BC-2059), a disruptor of the nuclear TBL1/R1-β-catenin-TCF7L2 complex represses c-Myc, cyclin D1 and Survivin and inhibits growth and survival of AML LSCs. In present studies, a CRISPR screen in UCSD-AML1 cells (with 3q26 lesion and EVI1 overexpression) with gRNAs targeting epigenetic regulators highlighted BRD4 and p300 as dependencies. Treatment with BETi, e.g., OTX015, dose-dependently induced apoptosis of AML cell lines and patient-derived (PD) AML cells (AML191 and AML194) with inv(3) or t(3;3). OTX015 treatment repressed c-Myc, c-Myb, CDK4, MECOM, and MCL1, while inducing HEXIM1 and cleaved PARP levels. TV treatment also dose-dependently induced apoptosis in AML cell lines and PD AML cells with 3q26.2 lesions, associated with depletion of nuclear β-catenin, EVI1, TCF7L2, c-Myc, c-Myb, RUNX1, CEBPα, c-KIT, BCL2, Bcl-xL and MCL1, but increase in p21, CD11b, BIM and cleaved PARP levels. Hi-ChIP with H3K27Ac antibody demonstrated that TV treatment abolished chromatin loops between MYC and its Es within the super-E. RNA-Seq analysis showed negative enrichment (log2-fold) of gene sets of MYC, E2F and WNT targets, DNA replication/repair and reduction of the 17-gene stemness score. Confocal microscopy showed that TV treatment disrupted co-localization of EVI1 and β-catenin with TBL1, also confirmed by the Proximity Ligation Assay. CyTOF analysis confirmed that TV treatment reduced EVI1, c-Myc, RUNX1, β-catenin, Bcl-xL, BCL2, MCL1 and Ki67 but increased cleaved PARP levels in the phenotypically characterized LSCs (with high expression of CLEC12A, CD123, CD244 and CD99) harboring inv(3) with EVI1 overexpression. Co-treatment with OTX015 and TV or the p300 inhibitor GNE-049 synergistically induced in vitro apoptosis in the AML cell lines and the PD AML cells with 3q26.2 lesions. Notably, in the flank-implanted PDX of AML191 or tail-vein infused PDX of AML242 models in NSG mice, treatment with OTX015 and TV, compared to vehicle control or each drug alone, significantly reduced AML growth and improved survival, without any host toxicity. These findings highlight the promising pre-clinical efficacy of novel BETi-based combinations against AML models harboring 3q26 lesions and EVI1 overexpression. Citation Format: Christine E. Birdwell, Warren C. Fiskus, Tapan M. Kadia, Christopher P. Mill, John A. Davis, Kaberi Das, Stephen Horrigan, Kapil N. Bhalla. Preclinical efficacy of targeting epigenetic mechanisms in AML with 3q26 lesions and EVI1 overexpression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4900.

Endogenous DOPA inhibits melanoma through suppression of CHRM1 signaling.

Melanoma risk is 30 times higher in people with lightly pigmented skin versus darkly pigmented skin. Using primary human melanocytes representing the full human skin pigment continuum and preclinical melanoma models, we show that cell-intrinsic differences between dark and light melanocytes regulate melanocyte proliferative capacity and susceptibility to malignant transformation, independent of melanin and ultraviolet exposure. These differences result from dihydroxyphenylalanine (DOPA), a melanin precursor synthesized at higher levels in melanocytes from darkly pigmented skin. We used both high-throughput pharmacologic and genetic in vivo CRISPR screens to determine that DOPA limits melanocyte and melanoma cell proliferation by inhibiting the muscarinic acetylcholine receptor M1 (CHRM1) signaling. Pharmacologic CHRM1 antagonism in melanoma leads to depletion of c-Myc and FOXM1, both of which are proliferation drivers associated with aggressive melanoma. In preclinical mouse melanoma models, pharmacologic inhibition of CHRM1 or FOXM1 inhibited tumor growth. CHRM1 and FOXM1 may be new therapeutic targets for melanoma.

653 Endogenous DOPA inhibits melanoma through suppression of CHRM1 signaling

Melanoma risk is 30 times higher in people with lightly pigmented skin compared to those with darkly pigmented skin. Here we show that this difference results from more than melanin pigment and its associated ultraviolet radiation (UVR) shielding effect. Using primary human melanocytes representing the full human skin pigment continuum and several preclinical melanoma models, we show that cell-intrinsic differences between dark and light melanocytes regulate melanocyte proliferative capacity, overall cellular differentiation state, and susceptibility to malignant transformation, independently of melanin and UV exposure. We determined that these differences result from dihydroxyphenylalanine (DOPA), a melanin precursor synthesized at higher levels in melanocytes from dark skin. Although DOPA was not previously known to have specific signaling activity, we used both high throughput pharmacologic and genetic in vivo CRISPR screens to determine that DOPA limits melanocyte and melanoma cell proliferation by inhibiting the muscarinic acetylcholine receptor M1 (CHRM1), a G Protein-Coupled Receptor (GPCR) not previously known to interact with DOPA, nor to affect melanoma pathobiology. Pharmacologic CHRM1 antagonism in melanoma leads to depletion of c-Myc and FOXM1, both of which are proliferation drivers associated with aggressive melanoma. In preclinical mouse melanoma models using both immune deficient and syngeneic immune competent mice, pharmacologic inhibition of CHRM1 or FOXM1 inhibited tumor growth. CHRM1 and FOXM1 may be new therapeutic targets for melanoma.

E2F1-induced lncRNA, EMSLR regulates lncRNA LncPRESS1

E2F1 induces hundreds of protein-coding genes influencing diverse signaling pathways but much less is known about its non-coding RNA targets. For identifying E2F1-dependent oncogenic long non-coding RNAs (lncRNAs), we carried out genome-wide transcriptome analysis and discovered an lncRNA, EMSLR, which is induced both in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). EMSLR depletion blocks the cells in G1 phase and inhibits the clonogenic ability indicating that it is essential for the tumor-related phenotypes. We discovered that EMSLR represses the promoter activity of another lncRNA, LncPRESS1, which is located 6.9 kb upstream of EMSLR and they display an inverse expression pattern in lung cancer cell lines. Depletion of C-MYC results in downregulation of EMSLR and simultaneous upregulation of EMSLR target LncPRESS1, exemplifying how C-MYC and E2F1 signal transduction pathways control the network of lncRNA genes to modulate cell proliferation and differentiation.

TP53 Mutations and RNA Binding Protein Musashi 2 Drive Resistance to PRMT5-Targeted Therapy in B-Cell Lymphoma

PRMT5 catalyzes the arginine symmetric demethylation of histone and non-histone proteins. PRMT5 is overexpressed in lymphoma and plays a key role in lymphomagenesis through the repression of several tumor suppressors, including P53, while promoting the expression of lymphoma-drivers, such as c-MYC.Currently, three PRMT5 inhibitors are being clinically evaluated in B-cell lymphoma patients, however, the mechanism of action of these molecules remain poorly understood. Thus, to identify the mechanism of action of the potent selective PRMT5 inhibitor, GSK3203591 (GSK-591), we performed a genome-wide CRISPR/Cas9 KO screen in the human mantle cell line, Z-138 and a validation CRISPR /Cas9 screen in the DLBCL cell line, OCI-LY19. Our genome-wide CRISPR screen identified 316 sensitizing genes and 89 genes involved in resistance. The top sensitizing gene was TP53, which validated our screens, as it is a well characterized target of PRMT5. Furthermore, we found that TP53 deletion and the hot spot TP53 R248Wmutation are biomarkers of resistance to GSK-591.RNA-binding protein MUSASHI 2 (MSI2) was identified as the top-ranked driver of resistance to GSK-591. MSI2 plays a key role in hematopoietic stem cells activation, myeloid leukemia and CLL, however, its function in lymphoma remains unknown. MSI2 is overexpressed in DLBCL patients (n=96). Relapsed MCL and DLBCL primary samples express high levels of both PRMT5 and MSI2. Furthermore, MSI2 depletion decreased cell viability and sensitized lymphoma cells to PRMT5 inhibitor in vitro and vivo. In contrast, overexpression of MSI2 reverted sensitivity to GSK-591 in lymphoma cells. Consistent with our genetic studies, inhibition of MSI2 using Ro 08-2750 (Ro) (Minuesa et al. Nat Commun. 2019), conferred sensitivity to GSK-591, and the combination therapy was synergistic inducing cytotoxicity.In order to identify specific downstream targets of MSI2 that may contribute to resistance to PRMT5 inhibition in lymphoma, we performed MSI2-HyperTRIBE, a recently discovered technology that allows mapping the MSI2 targeting network (Nguyen et al. Nat Commun. 2020). Using MSI2-HyperTRIBE, we were able to identify MSI2 targets in lymphoma cells. Moreover, we found that Ro treatment significantly blocked MSI2 binding activity, while GSK-591 had no effect on MSI2 targets.To uncover the mechanism involved in the synergy of PRMT5 and MSI2 inhibitors, we performed RNA-sequencing of Z-138 cells treated with GSK-591, Ro or the combination. Gene set enrichment analysis (GSEA) demonstrated a loss in the c-MYC pathway upon drug combination.MSI2 RNA-IP assays showed that MSI2 binds c-MYC and this interaction is disrupted by the combination of GSK-591 and Ro. We observed that the combination of MSI2 and PRMT5 inhibitors does not affect c-MYC mRNA levels but, rather, controls translation. Interestingly, in the human B cell line P493-6 that express a conditional, tetracycline-regulated c-MYC, the depletion of c-MYC significantly increased the anti-proliferative activity of GSK-591 and Ro alone or in combination.To further investigate whether there are other functionally relevant downstream targets of MSI2 promoting resistance to PRMT5 inhibition, we overlapped the differentially expressed genes upon treatment with GSK-591 and Ro (RNA-Seq), the Ro-direct targets in lymphoma (MSI2-HyperTRIBE) and the resistance genes to PRMT5 inhibition (whole-genome CRISPR screen) and we identified the anti-apoptotic protein BCL-2 as a common target of the PRMT5-MSI2 axis. We established first that the combination of GSK-591 and Ro blocked the binding of MSI2 to BCL-2 mRNA. We also found that the drug combination significantly decreased BCL-2 mRNA and protein abundance. Furthermore, Z-138 and OCI-LY19 BCL-2 KO cells are more sensitive to GSK-591 and Ro alone or in combination. Using venetoclax, we found that BCL-2 inhibition enhanced GSK-591 activity in vitro and in vivo. Thus, these data confirmed that BCL-2 is a key driver of resistance to PRMT5 inhibition.Overall, our study uncovered a novel oncogenic axis, PRMT5/MSI2/c-MYC/BCL-2 that drives resistance to PRMT5-targeted therapy in lymphoma. We demonstrated that TP53 LOF and MSI2 expression could be used as biomarkers for patient stratification. Moreover, we proposed two novel drug combination strategies, with venetoclax or a MSI2 inhibitor, to be considered in further clinical studies with PRMT5 inhibitors. DisclosuresBarbash: GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Batlevi: Viatris: Current holder of individual stocks in a privately-held company; Juno/Celgene: Consultancy; Pfizer: Current holder of individual stocks in a privately-held company; Bayer: Research Funding; ADC Therapeutics: Consultancy; TG Therapeutics: Consultancy; Medscape: Honoraria; BMS: Current holder of individual stocks in a privately-held company; Memorial Sloan Kettering Cancer Center: Current Employment; Kite Pharma: Consultancy; Life Sciences: Consultancy; Seattle Genetics: Consultancy; Karyopharm: Consultancy; Regeneron: Current holder of individual stocks in a privately-held company; Moderna: Current holder of individual stocks in a privately-held company; TouchIME: Honoraria; GLG Pharma: Consultancy; Dava Oncology: Honoraria; Xynomic: Research Funding; Roche/Genentech: Research Funding; Novartis: Research Funding; Epizyme: Research Funding; Janssen: Research Funding; Autolus: Research Funding. Younes: AZ: Current Employment, Other: Senior Vice President, Global Head of Haematology (Early and Late Stage) Oncology R&D at AstraZeneca.

Hsa_circ_0068631 promotes breast cancer progression through c-Myc by binding to EIF4A3

Breast cancer (BC) is one of the most common malignancies among women worldwide with a high incidence of recurrence and metastasis. In this study, we demonstrate that hsa_circ_0068631, a circRNA generated from the transferrin receptor (TFRC), is upregulated in BC tissues and cell lines. Knockdown of hsa_circ_0068631 inhibited the proliferation and migration of BC cells in vitro and in vivo. Mechanistically, an RNA pull-down assay and RNA immunoprecipitation assay revealed that eukaryotic translation initiation factor 4A3 (EIF4A3) could bind to hsa_circ_0068631 and c-Myc mRNA. Additionally, the expression of hsa_circ_0068631 was positively correlated with c-Myc, and the upregulation of hsa_circ_0068631 was a crucial factor for the dysregulation of c-Myc. Through an actinomycin D assay, we confirmed that the mRNA stability of c-Myc was influenced by hsa_circ_0068631 and EIF4A3. Furthermore, hsa_circ_0068631 could recruit EIF4A3 to increase c-Myc mRNA stability. Rescue assays manifesting depletion of c-Myc rescued the promotive effect of hsa_circ_0068631 overexpression on biological activities in BC. In conclusion, to our knowledge, this study is the first to unveil the role of hsa_circ_0068631 and the hsa_circ_0068631/EIF4A3/c-Myc axis in BC, providing a new target for BC treatment.

Vitamin K3 (menadione) Induces Cytotoxicity in Chronic Myeloid Leukemia Stem Cells By Upregulating DYRK2 through the Inhibition of SIAH2 Ubiquitin Ligase

Leukemic stem cells (LSCs) generated by the transformation of normal hematopoietic stem cells (HSCs) by BCR-ABL are elusive targets that can initiate and sustain leukemia owing to their unique capacity to regenerate themselves during cell division (self-renewal) and differentiate into a bulk of leukemia cells. Although chronic myeloid leukemia (CML) can be successfully managed using tyrosine kinase inhibitors (TKIs), drug discontinuation trials have shown that 30-60% of patients, depending on the drug used for first- and second-line treatment, can remain in treatment-free remission. Thus, discontinuation is still considered experimental and there are not safety guidelines to stop treatment. A better understanding of self-renewal mechanisms in LSCs will support the development of LSC-specific drugs to induce treatment-free remission in chronic-phase patients by eradicating BCR-ABL-positive LSCs. We recently discovered in mouse models that deletion of the Klf4 gene encoding for the transcription factor KLF4 severely abrogated progression of BCR-ABL(p210)-induced CML by impairing survival and self-renewal of LSCs but not normal hematopoietic stem cells (HSCs). Mechanistically, KLF4 represses the Dyrk2 gene and thus loss-of-KLF4 resulted in elevated DYRK2 protein levels, which was associated with p53-mediated apoptosis and inhibition of self-renewal via depletion of c-Myc protein in LSCs and not in normal HSCs. Here, we report that this mechanism is active in bone marrow cells from chronic-phase CML patients and that stabilization of DYRK2 protein by inhibition of the ubiquitin E3 ligase with vitamin K3 (VK3) has the potential therapeutic approach. Treatment of CML cell lines and bone marrow cells from CML patients with VK3 induced dose-dependent cytotoxicity and apoptosis. This was correlated with increased DYRK2 expression, depletion of c-Myc, and activation of p53-mediated apoptosis. Most importantly, VK3 induced DYRK2 expression and abrogated capacity of purified CD34+ CML stem cells, but not normal CD34+ cells, to generate colonies in methylcellulose assays. Cytotoxicity of VK3 largely depended on the expression of DYRK2, the reduced form menadiol inhibited cell viability similarly to menadione (VK3), and the presence of p53 enhanced cytotoxicity by inducing apoptosis in addition to inhibition of self-renewal. VK3 augmented cytotoxicity of Imatinib in CML cell lines and inhibited cell survival in Imatinib-resistant CML cells. The ability to target CML LSCs was confirmed by in vivo administration of VK3 in BCR-ABL induced mouse model followed by transplantation of leukemic bone marrow in secondary recipient mice. Altogether, our results suggest that stabilization of DYRK2 by inhibition of SIAH2 can induce apoptosis in bulk leukemia cells and LSCs by activating p53 and depleting c-Myc. DisclosuresNo relevant conflicts of interest to declare.

TRIM66 Overexpression Promotes Glioma Progression and Regulates Glucose Uptake Through cMyc/GLUT3 Signaling.

ObjectiveTripartite motif 66 (TRIM66) is reported to be closely associated with human cancers. However, the roles of TRIM66 in glioma remain unclear. The present study aimed to investigate the clinical significance and biological roles of TRIM66 in human glioma.MethodsTRIM66 expression in glioma tissues was examined by immunohistochemistry. TRIM66 overexpression and siRNA knockdown were performed in glioblastoma cell lines. CCK8, colony formation assay, transwell assay, Annexin V and JC1 staining, glucose uptake assay, and Western blotting were used to explore the biological roles and potential underlying mechanisms of TRIM66 in glioma progression.ResultsOur results showed that TRIM66 was overexpressed in 52/95 glioma cases. The rates of TRIM66 overexpression in Grade I, Grade II, Grade III, and Grade IV gliomas were 16.6%, 41.3%, 58.6%, and 70.9%, respectively. Oncomine data showed that TRIM66 was upregulated in glioblastoma and oligodendroglioma compared with normal brain tissues. TRIM66 expression was higher in glioblastoma cell lines compared with normal SVG p12 glial cell line. TRIM66 promoted in vitro and in vivo proliferation, invasion, and inhibited temozolomide (TMZ)-induced apoptosis. Notably, TRIM66 increased glucose metabolism by upregulating glucose uptake, glucose consumption, and ATP production. Western blotting showed that TRIM66 positively regulated cMyc and GLUT3. Depletion of cMyc by siRNA abolished the effect of TRIM66 on GLUT3. Chromatin immunoprecipitation (ChIP) assay showed that cMyc could bind to the promoter regions of GLUT3 in glioblastoma cells.ConclusionTRIM66 was upregulated in human gliomas, where it promoted cell growth and chemoresistance. Our data also identified novel roles of TRIM66 in glioma progression. TRIM66 upregulates glucose uptake and mitochondrial function through the cMyc/GLUT3 signaling, which makes it a potential therapeutic target.

Valtrate as a novel therapeutic agent exhibits potent anti-pancreatic cancer activity by inhibiting Stat3 signaling

BackgroundValtrate is a novel epoxy iridoid ester isolated from Chinese herbal medicine Valeriana jatamansi Jones with anti-proliferative activity against various human cancer cell lines. However, its efficacy and molecular mechanisms against pancreatic cancer (PC) cells are largely unclear. PurposeTo investigate the anti-cancer effects of valtrate on PC cell lines and its underlying mechanisms. MethodsMTT assay was first performed to detect the effect of valtrate on cell viability in human PC cell lines and normal pancreatic epithelial cells HPDE. Cell apoptosis and cycle phase assay were detected by flow cytometry. The relative mRNA expressions of Bax, Bcl-2, c-Myc, and CyclinB1 were tested by quantitative PCR (qPCR) assay. The expression of relative proteins was detected by Western blotting (WB). A PANC-1luc cells xenograft mouse model in nu/nu female mice was used to elucidate the effect of valtrate on tumor growth in vivo. ResultsValtrate significantly inhibited the growth of PC cells without affecting the growth of normal pancreatic epithelial cells HPDE, induced significant apoptosis and cell cycle arrest in G2/M phase. Moreover, valtrate inhibited the tumor growth of PC cell PANC-1 in xenograft mice by 61%. Further mechanism study demonstrated that valtrate could increase the expression level of Bax, suppress Bcl-2 as well as c-Myc and Cyclin B1, inhibit the transcriptional activity of Stat3, while valtrate decreased the expression level of Stat3 and phosphated-Stat3 (Tyr705) and induced the high molecular aggregation of Stat3. Molecular docking analysis predicted that valtrate might interact with Cys712 of Stat3 protein. Valtrate could also induce a transient depleted intracellular glutathione (GSH) level and increased reactive oxygen species (ROS). NAC (N-acetylcysteine), a reducer reversed valtrate-induced the depletion of Stat3, p-Stat3, c-Myc, and Cyclin B1. ConclusionValtrate exerts anti-cancer activity against PC cells by directly targeting Stat3 through a covalent linkage to inhibit Stat3 activity, which causes apoptosis and cell cycle arrest.

Metabolic sensor O-GlcNAcylation regulates megakaryopoiesis and thrombopoiesis through c-Myc stabilization and integrin perturbation.

Metabolic state of hematopoietic stem cells (HSCs) is an important regulator of self‐renewal and lineage‐specific differentiation. Posttranslational modification of proteins via O‐GlcNAcylation is an ideal metabolic sensor, but how it contributes to megakaryopoiesis and thrombopoiesis remains unknown. Here, we reveal for the first time that cellular O‐GlcNAcylation levels decline along the course of megakaryocyte (MK) differentiation from human‐derived hematopoietic stem and progenitor cells (HSPCs). Inhibition of O‐GlcNAc transferase (OGT) that catalyzes O‐GlcNAcylation prolongedly decreases O‐GlcNAcylation and induces the acquisition of CD34+CD41a+ MK‐like progenitors and its progeny CD34−CD41a+/CD42b+ megakaryoblasts (MBs)/MKs from HSPCs, consequently resulting in increased CD41a+ and CD42b+ platelets. Using correlation and co‐immunoprecipitation analyses, we further identify c‐Myc as a direct downstream target of O‐GlcNAcylation in MBs/MKs and provide compelling evidence on the regulation of platelets by novel O‐GlcNAc/c‐Myc axis. Our data indicate that O‐GlcNAcylation posttranslationally regulates c‐Myc stability by interfering with its ubiquitin‐mediated proteasomal degradation. Depletion of c‐Myc upon inhibition of OGT promotes platelet formation in part through the perturbation of cell adhesion molecules, that is, integrin‐α4 and integrin‐β7, as advised by gene ontology and enrichment analysis for RNA sequencing and validated herein. Together, our findings provide a novel basic knowledge on the regulatory role of O‐GlcNAcylation in megakaryopoiesis and thrombopoiesis that could be important in understanding hematologic disorders whose etiology are related to impaired platelet production and may have clinical applications toward an ex vivo platelet production for transfusion.

A cell competition–based small molecule screen identifies a novel compound that induces dual c-Myc depletion and p53 activation

Breakpoint Cluster Region-Abelson kinase (BCR–Abl) is a driver oncogene that causes chronic myeloid leukemia and a subset of acute lymphoid leukemias. Although tyrosine kinase inhibitors provide an effective treatment for these diseases, they generally do not kill leukemic stem cells (LSCs), the cancer-initiating cells that compete with normal hematopoietic stem cells for the bone marrow niche. New strategies to target cancers driven by BCR–Abl are therefore urgently needed. We performed a small molecule screen based on competition between isogenic untransformed cells and BCR–Abl-transformed cells and identified several compounds that selectively impair the fitness of BCR–Abl-transformed cells. Interestingly, systems-level analysis of one of these novel compounds, DJ34, revealed that it induced depletion of c-Myc and activation of p53. DJ34-mediated c-Myc depletion occurred in a wide range of tumor cell types, including lymphoma, lung, glioblastoma, breast cancer, and several forms of leukemia, with primary LSCs being particularly sensitive to DJ34. Further analyses revealed that DJ34 interferes with c-Myc synthesis at the level of transcription, and we provide data showing that DJ34 is a DNA intercalator and topoisomerase II inhibitor. Physiologically, DJ34 induced apoptosis, cell cycle arrest, and cell differentiation. Taken together, we have identified a novel compound that dually targets c-Myc and p53 in a wide variety of cancers, and with particularly strong activity against LSCs.

BET Protein Antagonist-Based Therapy of Novel Models of Richter Transformation-Diffuse Large B-Cell Lymphoma (RT-DLBCL)

Richter Transformation (RT) is the development of aggressive DLBCL (mostly ABC sub-type) in up to ~15% of patients with antecedent CLL. Approximately 80% of RT-DLBCLs are clonally-related (CLR) to the underlying CLL, with a median survival (MS) of one year. Alternatively, ~20% of RT-DLBCLs are clonally-unrelated (CLUR) to the underlying CLL, exhibiting a better MS of 5 years. Chemo-immunotherapy, or monotherapy with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, anti-apoptotic BCL2 inhibitor venetoclax or with anti-PD1 therapy fails to achieve prolonged disease-free survival, with a majority relapsing with therapy-refractory disease. To develop and test novel targeted therapies for RT-DLBCL, we successfully established three patient-derived xenograft (PDX) models of luciferized RT-DLBCLs, including the CLR HPRT3 and CLUR HPRT2. HPRT3 and HPRT2 cells were of ABC sub-type of DLBCL, based on positive MUM1/IRF4 and negative CD10 and BCL6 expressions. The third RT-DLBCL (HPRT1) was a rarer GCB variety of DLBCL, displaying high CD10, BCL6, Ki-67 and c-Myc expressions. FISH analysis confirmed 5' MYC amplification in HPRT1 cells. Cytogenetic and array-CGH analyses showed large numbers of karyotypic and genetic alterations in HPRT3 and HPRT2 RT-DLBCLs. Low-pass whole genome sequencing showed that HPRT3 and HPRT2, but not HPRT1, exhibit large areas of amplification of DNA on chromosome 18, with copy gains at 18q21.1 involving the TCF4 gene. NextGen sequencing (NGS) of a panel of 300 genes (L-300 panel) showed more genetic mutations (at high % VAF) in HPRT3, as compared to HPRT2 or HPRT1 cells. These genetic alterations targeted transcription factors, epigenetic regulators, DNA damage/repair enzymes, signaling enzymes and their regulators. Utilizing ATAC-Seq, ChIP-Seq with H3K27Ac and BRD4 antibodies, and single cell (sc) RNA-Seq analyses, we evaluated active chromatin and gene-expressions in the three RT-DLBCLs. ChIP-Seq-determined signal-density of H3K27Ac and BRD4, as well as ATAC-Seq-determined peak-density were increased at active SE/E of BCL2, TCF4, PAX5 and IRF4 in HPRT3 and HPRT2 cells. Notably MYC, BCL6 and CDK6 SEs were active in HPRT1 cells. scRNA-Seq generated t-SNE plots showed that HPRT3 cells exhibited highest number of transcriptionally active cell-clusters, with high mRNA expressions of TCF4, PAX5 and IRF4 in HPRT3 and HPRT2 cells. HPRT1 cells overexpressed MYC mRNA. HPRT3 and HPRT2 were relatively more sensitive to venetoclax-induced lethality, which correlated with higher BCL2, BAX, BAK and BIM protein levels. Higher Bcl-xL levels correlated with increased sensitivity of HPRT3 and HPRT2 versus HPRT1 cells to a Bcl-xL-specific inhibitor A-1155463. Conversely, higher MCL1 levels correlated with greater sensitivity of HPRT1 cells to an MCL1-specific inhibitor AZD-5991. Notably, HPRT3 and HPRT2 versus HPRT1 cells were relatively resistant to ibrutinib treatment. This was due to activation of the alternative MAP3K14 (NIK kinase)-NFkB pathway with increased processing of p100 to p52. Treatment with CDK9 inhibitor NVP2 dose-dependently induced apoptosis of the 3 RT-DLBCL cells, associated with depletion of c-Myc, Bcl-xL, and MCL1 protein levels. BET inhibitor OTX015 and BET protein degrader ARV-771 induced more lethality in HPRT1, compared to HPRT3 and HPRT2 cells, which correlated with higher levels of BRD4, c-Myc and TCF4, but markedly lower levels of IRF4 in HPRT1 cells. CRISPR-Cas9-mediated knockout (KO) of IRF4 markedly depleted nuclear levels of IRF4 and c-Myc (a target of IRF4), which significantly increased sensitivity to OTX015 in IRF4 KO RT-DLBCL cells. Co-treatment with OTX015 and ibrutinib or venetoclax was synergistically lethal in all three RT-DLBCL cell-types (CI < 1.0). Co-treatment with BET protein degrader ARV-771 and ibrutinib or venetoclax exerted synergistic lethality in all three RT-DLBCL cell-types (CI < 1.0). Finally, following tail vein infusion and engraftment of HPRT3 cells, daily treatment for three weeks with ARV-771 and venetoclax compared to each drug or vehicle control, significantly reduced the RT-DLBCL burden, as well as improved survival without inducing toxicity in the NSG mice. These findings strongly support further testing of BET protein antagonist-based combinations with BH3 mimetics, BTK inhibitors, as well as with other novel agents, utilizing pre-clinical models of RT-DLBCL. Disclosures Green: KDAc Therapeutics: Current equity holder in private company.

Preclinical Efficacy of a Novel Omacetaxine-Based Therapy for AML Expressing Somatic or Germline Mutant RUNX1

RUNX1 is the DNA-binding subunit of the core-binding factor complex and a master-regulator transcription factor (TF), which is involved in normal and malignant hematopoiesis. Somatic mutations in RUNX1 commonly occur in Myelodysplastic Syndromes (MDS), as well as in secondary (s) or de novo AML (~10%). The germline, intragenic mutations and deletions in RUNX1 cause the highly penetrant (~40%), autosomal dominant, Familial Platelet Disorder (RUNX1-FPD) by the median age of 33 years with a propensity to evolve into myeloid malignancy (FPD-MM), i.e., MDS or AML. Majority of mutant (mt) RUNX1 are missense, large deletions or truncation-mutations, behaving mostly as loss of function (LOF) mutations. Lack of specific targeted therapy, coupled with resistance to standard chemotherapy, may account for poorer outcome observed in AML or FPD-MM expressing somatic or germline mutant (mt) RUNX1, respectively. We previously reported that knockdown (KD) of RUNX1 (wild type and mtRUNX1) is significantly more lethal toward AML blasts progenitor cells (BPCs) expressing mt versus wild type (wt) RUNX1 or normal hematopoietic progenitor cells (HPCs), indicating greater dependency on the residual wtRUNX1. Based on higher occupancy of the BET protein BRD4 at the RUNX1 super-enhancer/enhancer (SE/E) and other SE-driven AML-relevant oncogenes, the BET inhibitor OTX015 evicted BRD4 from the chromatin and repressed RUNX1 and SE-driven oncogenes, mediating efficacy against AML BPCs expressing mtRUNX1. Utilizing the RNA-Seq signature of RUNX1-depleted (by shRNA) AML cells and conducting LINCS (Library of Integrated Network-based Cellular Signatures) 1000-CMap (Connectivity Mapping) analysis, we discovered several expression mimickers (EMs), including the top-ranking protein-translation inhibitor homoharringtonine (HHT), or its semi-synthetic analogue omacetaxine mepesuccinate (OM). We tested the pre-clinical efficacy of HHT or OM against AML cell lines (OCI-AML5 and Mono-Mac-1) and patient-derived primary AML BPCs expressing somatic mtRUNX1 or wtRUNX1. Treatment with HHT or OM (5 to 100 nM) induced significantly more lethality in OCI-AML5 and Mono-Mac-1, as well as primary AML BPCs expressing mutant versus wtRUNX1, or in normal bone marrow progenitor cells (p < 0.05). Notably, first-time ever, CRISPR-Cas9-mediated knock-in of mtRUNX1 (R174*) into wtRUNX1-expressing AML HL-60 and OCI-AML2 cells showed that, in this isogenic setting also, HHT or OM, or OTX015 (50 to 1000 nM) treatment induced significantly more lethality in mt versus wtRUNX1-expressing cells (p < 0.05). The differential lethal activity of HHT in cells was associated with marked HHT-mediated depletion of c-Myc, c-Myb, PU.1, mt/wtRUNX1 and MCL1 in mtRUNX1 expressing HL60-R174* and OCI-AML2-R174* cells. Possibly due to HHT-mediated MCL1 repression, co-treatment with HHT and venetoclax (V) (10 to 100 nM), synergistically induced in vitro apoptosis of OCI-AML5, HL60-R174* and OCI-AML2-R174* cells (CI < 1.0). Compared to vehicle control, whereas daily treatment with OM (1.0 mg/kg, SQ) alone for 3 weeks significantly reduced the leukemia burden, co-treatment with OM and V (20 mg/kg orally) exerted significantly greater reduction in AML burden due to luciferized OCI-AML2-R174* cells engrafted into NSG mice. Combined therapy with OM and V also improved survival of the mice engrafted with OCI-AML2-R174* cells, without inducing weight loss or any other toxicity. Treatment with HHT also exerted differentially greater in vitro lethality against HPCs from FPD-MM expressing germline mtRUNX1 along with other MDS/AML-associated co-mutations, compared to RUNX1-FPD HPCs. We recently also established in culture, first time ever, a germ-line mtRUNX1 (K194N)-expressing FPD/MM cell line (GMR-AML1) from the bone marrow aspirate cells of a patient with FPD-MM that also expressed mutations in BCOR, PHF6, SRFS2 and SF3B1. Exposure to HHT (10 to 100 nM) dose-dependently inhibited cell growth and induced lethality in GMR-AML1 cells. These studies highlight that treatment with OM could potentially revert hematopoiesis in FPD-MM back to the RUNX1-FPD status. Collectively, our preliminary findings strongly support the merit of testing co-treatment with OM and venetoclax in patients with AML expressing somatic mtRUNX1, as well as in FPD-MM prior to or instead of allogeneic SCT from an unrelated donor. Disclosures DiNardo: Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Syros: Honoraria; Jazz: Honoraria; Agios: Consultancy, Honoraria, Research Funding; ImmuneOnc: Honoraria; MedImmune: Honoraria; Calithera: Research Funding; Notable Labs: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Novartis: Consultancy.

Abstract 4849: YPN-005, an oral CDK7 inhibitor, shows a significant antitumor activity in Myc amplified solid tumors and MCL-1 overexpressed blood cancer

Abstract Background: Cyclin dependent kinase 7 (CDK7) has been recognized as one of potential therapeutic targets in Myc-driven cancers. Previously, we synthesized selective irreversible CDK7 inhibitor and we demonstrated a significant inhibition of the proliferation of TNBC and HCC cells by down-regulation of c-Myc and MCL-1. We further report YPN-005 of the preclinical data with biological characterization. Methods: Antiproliferative activity of YPN-005 was measured in solid tumor cell lines with Myc amplification and in hematologic cancer cells with MCL-1overexpression. To investigate the mechanism of action of YPN-005, the expression levels of phospho-RNA polymerase II, phospho-CDK2 and -CDK4, BClXL, cleaved-PARP, c-Myc and MCL-1 were analyzed by Western Blot analyses. CDK7 occupancy was determined by using a biotinylated small molecule probe following incubating of TNBC, HCC, AML cells and hPBMC with YPN-005. The therapeutic efficacy of YPN005 was evaluated in TNBC and HCC xenograft mouse model. Results: YPN-005 treatment induced apoptosis and cell cycle arrest. Selectivity of YPN-005 on CDK7 was confirmed in the 468 kinases panel assay. YPN-005 significantly inhibited the proliferation of TNBC, ER+ breast cancer, HCC, ovarian cancer, small cell lung cancer (SCLC), pancreas, prostate and various types of blood cancer cells and IC50's were determined in 1-20 nM range. Inhibition of cell proliferation was accompanied by down-regulation of RNA PolII phosphorylation, c-Myc and MCL-1 expression. Washout assay by Western Blot demonstrated that YPN-005 irreversibly blocked RNA PolII phosphorylation. In addition, YPN-005 downregulated the adaptive immune checkpoint PD-L1 by the depletion of c-Myc in TNBCs. CDK7 occupancy over 90% was observed in TNBC, HCC, and AML cells with 100nM of YPN-005. In vivo xenograft models of TNBC and HCC showed that daily oral administration of YPN-005 for 21 days (once, and 10~20mg/kg) efficiently inhibited the growth of tumor. All the mice survived and there was no significant changes in their hematologic profiles. Conclusion: Our findings suggest that YPN-005 is an orally available, potent, and selective CDK7 inhibitor which can be used for the treatment of Myc and MCL-1 overexpressing cancer patients. These data support the rationale for advancing YPN-005 towards IND-enabling studies. Citation Format: Jakyung Yoo, Jun Hee Lee, Mi Jung Lee, Kang Woo Lee, Ji Eun Min, Jinhwan Kim, Ki-Nam Min, Da-Hye Jeon, Dae Seong Lim, Kwang-Ok Lee. YPN-005, an oral CDK7 inhibitor, shows a significant antitumor activity in Myc amplified solid tumors and MCL-1 overexpressed blood cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4849.

Role of B Cell Lymphoma 2 in the Regulation of Liver Fibrosis in miR-122 Knockout Mice.

MicroRNA-122 (miR-122) has been identified as a marker of various liver injuries, including hepatitis- virus-infection-, alcoholic-, and non-alcoholic steatohepatitis (NASH)-induced liver fibrosis. Here, we report that the extracellular miR-122 from hepatic cells can be delivered to hepatic stellate cells (HSCs) to modulate their proliferation and gene expression. Our published Argonaute crosslinking immunoprecipitation (Ago-CLIP) data identified several pro-fibrotic genes, including Ctgf, as miR-122 targets in mice livers. However, treating Ctgf as a therapeutic target failed to rescue the fibrosis developed in the miR-122 knockout livers. Alternatively, we compared the published datasets of human cirrhotic livers and miR-122 KO livers, which revealed upregulation of BCL2, suggesting its potential role in regulating fibrosis. Notably, ectopic miR-122 expression inhibited BCL2 expression in human HSC (LX-2) cells). Publicly available ChIP-seq data in human hepatocellular cancer (HepG2) cells and mice livers suggested miR-122 could regulate BCL2 expression indirectly through c-MYC, which was confirmed by siRNA-mediated depletion of c-MYC in Hepatocellular Carcinoma (HCC) cell lines. Importantly, Venetoclax, a potent BCL2 inhibitor approved for the treatment of leukemia, showed promising anti-fibrotic effects in miR-122 knockout mice. Collectively, our data demonstrate that miR-122 suppresses liver fibrosis and implicates anti-fibrotic potential of Venetoclax.

Efficacy of Targeted Therapy in Novel Pre-Clinical in Vitro and In Vivo Models of Richter Transformation-Diffuse Large B-Cell Lymphoma (RT-DLBCL)

Richter Transformation (RT) is defined as the development of aggressive DLBCL (mostly ABC-type) in up to ~15% of patients with antecedent or concurrent diagnosis of CLL. Based on the comparison of immunoglobin gene rearrangements, approximately 80% of RT-DLBCL arise due to a direct clonal evolution of the underlying CLL clone, i.e., clonally related (CLR) RT-DLBCL, which exhibit poor median survival (MS) of one year. Approximately 20% of RT-DLBCLs are clonally unrelated (CUR) to the underlying CLL, arising most likely due to branched clonal evolution from a common pre-CLL progenitor. CUR RT-DLBCLs exhibit a better MS of 5 years. Although chemo-immunotherapy and treatment with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib or anti-apoptotic BCL2 inhibitor venetoclax can induce remissions, they fail to induce prolonged disease-free survival in RT-DLBCL. Majority of patients relapse with therapy-refractory disease. Lack of availability of in vitro cultured RT-DLBCL cells or PD xenograft models has prevented pre-clinical testing and development of novel targeted agents against RT-DLBCL. Here, we report the establishment of 3 patient-derived xenograft (PDX) models of RT-DLBCL. Based on immunoglobulin heavy chain (IGH) clonality testing by NGS, the RT-DLBCL RT17 was CLR, RT15 was CUR and RT5 was of indeterminate clonality. The PDXs were generated by tail-vein infusion and engraftment of luciferase-transduced CD19+ RT-DLBCL cells in NSG mice. The RT-DLBCL PDXs grew in the bone marrow and spleen, causing marked splenomegaly, requiring euthanasia 4 to 6 weeks after engraftment. All three RT-DLBCL PDX cells were EBV-negative by genomic and EBNA2 protein expression analyses. NextGen DNA sequencing of RT17, RT15, and RT5 cells showed large numbers of genetic mutations, including mutations in TP53, ATM, NOTCH2, TET2 and MLL3 genes with a high variant allelic frequencies. Array-CGH showed DNA copy gains or losses in multiple chromosomes, including 3, 8, 9, 11, 12, 17 and 18. A 5'-MYC amplification was detected by FISH analysis in RT5 DLBCL cells. ATAC-Seq showed increased signal intensity representing increased chromatin accessibility in the RT-DLBCL cells compared to CD34+ normal progenitors. High peak numbers were detected in specific loci, including TCF4, PAX5, IRF4, MYB, MYC, BCL2L1 and BCL-2. Anti-H3K27Ac ChIP-Seq analysis showed increased average, normalized read-densities at super-enhancers/enhancers (SEs/Es), including those of TCF4, PAX5, IRF4, BCL2 (RT17 and RT15) and MYC (RT5). Western analyses showed that all three RT-DLBCL PDX cells expressed TCF4, c-Myc, and BRD4, with highest expression in RT5 cells. Accordingly, RT5 cells were more sensitive than cells RT17 and RT15 cells to the BET protein inhibitor (BETi) OTX015-induced apoptosis. This was associated with greater, OTX015-mediated, depletion of c-Myc in RT5 cells. RT17 and RT15 expressed high levels of BCL2, Bcl-xL and MCL1, whereas RT5 lacked BCL2 expression. Consistent with this, RT17 and RT15 cells were significantly more sensitive than RT5 cells to venetoclax-induced apoptosis (p < 0.01). RT17 and RT15, but not RT5 cells, expressed NFkB2 (p52), consistent with activation of non-canonical NFkB signaling. This was associated with resistance of RT17 and RT15 cells to ibrutinib-induced apoptosis (p < 0.001). However, co-treatment with OTX015 and ibrutinib or venetoclax induced synergistic lethality in all RT-DLBCL cells (combination indices < 1.0). BET-PROTAC ARV-825 and ARV-771 treatment depleted BRD4, leading to marked reduction in c-Myc levels and apoptosis of all RT-DLBCL cells. Treatment with the ATP-competitive, CDK9 inhibitor NVP2 also dose-dependently induced apoptosis in RT-DLBCL cells associated with depletion of c-Myc, Bcl-xL, and MCL1 protein levels. These findings highlight the activity and support further in vitro and in vivo evaluation of BETi, BET-PROTAC or CDK9i-based combinations with ibrutinib or venetoclax against genetically-profiled RT-DLBCL cells that are clonally-related or clonally-unrelated to the antecedent CLL. Disclosures Maiti: Celgene: Other: research funding. Bhalla:Beta Cat Pharmaceuticals: Consultancy. Khoury:Angle: Research Funding; Stemline Therapeutics: Research Funding; Kiromic: Research Funding.

The Sustained Induction of c-MYC Drives Nab-Paclitaxel Resistance in Primary Pancreatic Ductal Carcinoma Cells.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with limited and, very often, ineffective medical and surgical therapeutic options. The treatment of patients with advanced unresectable PDAC is restricted to systemic chemotherapy, a therapeutic intervention to which most eventually develop resistance. Recently, nab-paclitaxel (n-PTX) has been added to the arsenal of first-line therapies, and the combination of gemcitabine and n-PTX has modestly prolonged median overall survival. However, patients almost invariably succumb to the disease, and little is known about the mechanisms underlying n-PTX resistance. Using the conditionally reprogrammed (CR) cell approach, we established and verified continuously growing cell cultures from treatment-naïve patients with PDAC. To study the mechanisms of primary drug resistance, nab-paclitaxel-resistant (n-PTX-R) cells were generated from primary cultures and drug resistance was verified in vivo, both in zebrafish and in athymic nude mouse xenograft models. Molecular analyses identified the sustained induction of c-MYC in the n-PTX-R cells. Depletion of c-MYC restored n-PTX sensitivity, as did treatment with either the MEK inhibitor, trametinib, or a small-molecule activator of protein phosphatase 2a. IMPLICATIONS: The strategies we have devised, including the patient-derived primary cells and the unique, drug-resistant isogenic cells, are rapid and easily applied in vitro and in vivo platforms to better understand the mechanisms of drug resistance and for defining effective therapeutic options on a patient by patient basis.