scholarly journals Resistance to SL-401 in AML and BPDCN Is Associated with Loss of the Diphthamide Synthesis Pathway Enzyme DPH1 and Is Reversible By Azacitidine

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 797-797 ◽  
Author(s):  
Jason Stephansky ◽  
Katsuhiro Togami ◽  
Mahmoud Ghandi ◽  
Joan Montero ◽  
Nick vonEgypt ◽  
...  
Keyword(s):  
Cell Lines ◽  
De Novo ◽  
Phase 1 ◽  
Acquired Resistance ◽  
Survival Advantage ◽  
Target Cells ◽  
Rna Seq ◽  
Adp Ribosylation ◽  
Synergistic Cytotoxicity ◽  
Resistant Cells

Abstract SL-401 is a novel targeted therapy comprised of recombinant interleukin 3 (IL3) fused to a truncated diphtheria toxin (DT) payload. SL-401 delivers DT to cells expressing the IL3 receptor (CD123). After internalization, DT catalyzes ADP ribosylation of eukaryotic elongation factor 2 (eEF2), blocking protein synthesis and killing target cells. SL-401 is currently in clinical trials for CD123+ cancers, including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Other than CD123 expression, the determinants of response are largely unknown. Our goal was to study mechanisms of de novo and acquired resistance to inform combination strategies and enhance the efficacy of SL-401. In 16 AML and BPDCN patients enrolled in a phase 1-2 trial, we did not see decreased expression of CD123 by flow cytometry during or after SL-401. To study alternative resistance mechanisms, we generated 3-6 independent SL-401 resistant clones from each of 4 CD123+ AML (THP1, NOMO1, EOL1) or BPDCN (CAL1) cell lines. We treated cells with the LD95 (lethal dose to 95%), retreating upon recovery. All lines developed >2-3 log resistance to SL-401 within 28 days. All resistant clones maintained CD123 surface expression, consistent with what we observed in patients. Using confocal microscopy, we saw that a fluorescently tagged SL-401 was internalized equally in resistant and parental cells. SL-401 resistant cells were also resistant to full length DT, suggesting that the mechanism of resistance involved DT rather than IL3 binding/internalization. We performed whole transcriptome RNA-sequencing and whole exome sequencing (WES) on parental and SL-401 resistant cells. There were no recurrent acquired DNA mutations. However, in RNA-seq the most downregulated gene in 6 independent clones from 2 lines was DPH1 (FC -7.5, FDR<0.0001). DPH1 is the first enzyme in a cascade that converts histidine 715 on eEF2 to diphthamide, the direct target for ADP ribosylation by DT. Decreased expression of DPH1 was confirmed in the 6 resistant clones by qRT-PCR, and in 3 clones from an additional line. Across 33 cell lines and subclones there was an inverse linear correlation between DPH1 level and SL-401 IC50 (P=0.0005). To validate this finding in patients, we performed paired RNA-seq on CD45+CD123+ sorted blasts from 2 AMLs pre & post 2 cycles of SL-401. Both patients' AMLs had reduced DPH1 after exposure to SL-401 (mean -2.1 fold). To determine if loss of DPH1 was sufficient to confer de novo resistance to SL-401, we generated THP1, NOMO1, and CAL1 cells stably expressing the Cas9 nuclease and transduced them with 1 of 4 DPH1-targeting or 2 non-targeting CRISPR sgRNAs co-expressing GFP. Four days after infection at titers that achieve ~20% GFP+ cells, we treated with the LD95 of SL-401. We observed a survival advantage for DPH1 sgRNA-transduced cells (2.9 fold increase in GFP+ cells, P<0.0001) and decreased apoptosis measured by AnnexinV positivity, compared to GFP- cells in the same cultures. By contrast, there was no survival advantage for cells transduced with control sgRNAs. These data suggest that DPH1 loss is sufficient to mediate SL-401 resistance in AML and BPDCN. Pseudomonas toxin (PT) also targets eEF2 on diphthamide, and PT resistance in a B-ALL cell line has been associated with DPH1 loss via reversible promoter CpG DNA methylation (Hu, Leuk Res 2013). Therefore, we tested the DNA methyltransferase inhibitor azacitidine in combination with SL-401 and observed synergistic cytotoxicity, in naïve (combination index (CI) = 0.45; <1 indicates synergy) and SL-401 resistant (CI = 0.55) cells. Most strikingly, 4-week pulsatile treatment with non-lethal "epigenetic" doses of azacitidine (300 nM 2d on/2d off) fully reversed SL-401 resistance in 6 CAL1 and THP1 clones that were insensitive at baseline (Figure). Controls grown in vehicle or with weekly SL-401 challenge showed no reversion, suggesting that azacitidine had a specific sensitizing effect. Restoration of SL-401 sensitivity was accompanied by an increase in DPH1 expression compared to resistant clones. In summary, we found that DPH1 is a biomarker of SL-401 activity and acquired resistance, and resistance is reversible by azacitidine. Based on these data, we have initiated a multicenter phase 1 trial of the combination of SL-401 and azacitidine in patients with AML or MDS (NCT03113643), with correlative laboratory studies designed to explore these hypotheses. Disclosures vonEgypt: Stemline Therapeutics: Employment. Lindsay: Stemline Therapeutics: Employment. Brooks: Stemline Therapeutics: Employment, Equity Ownership, Patents & Royalties. Lane: Stemline Therapeutics: Research Funding; N-of-one: Consultancy.

scholarly journals Activation of the ERK Pathway Drives Acquired Resistance to Venetoclax in MM Cell Models

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-22
Author(s):  
Chandraditya Chakraborty ◽  
Yan Xu ◽  
Yao Yao ◽  
Eugenio Morelli ◽  
Anil Aktas-Samur ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Acquired Resistance ◽  
Gene Set Enrichment Analysis ◽  
Erk Pathway ◽  
Rna Seq ◽  
Intrinsic Resistance ◽  
Private Company ◽  
Advisory Committees ◽  
High Doses

Multiple myeloma (MM) is a hematological malignancy characterized by various genetic abnormalities including translocations involving the IgH gene at 14q32. Amongst these, t(11;14) is one of the most common translocations. Recent clinical data suggests a significant impact of Venetoclax, a small molecule inhibitor of BCL2, in this subgroup of MM patients, representing the first example of personalized medicine in MM and opening a wide range of research aiming at elucidating its mechanism of action. However, despite the initial positive response to the drug, a significant proportion of patients eventually develop resistance and relapse. To delineate the mechanisms that contribute to the development of an acquired drug-tolerant/resistance phenotype, we modeled the response to Venetoclax in 2 MM cell lines (KMS27 and KMS-12PE with IC50 of 35.47nM and 3.64nM, respectively). Whereas the vast majority of cells plated into 96-well plates were killed within a few days of exposure to a high dose of drug concentration, we detected a small fraction of viable, largely quiescent cells, which were expanded by culturing them in high doses of Venetoclax. We successfully generated 4 independent clones from each cell line, that were single cell-cloned with continued growth in the presence of high doses of Venetoclax. These clones labelled as drug-tolerant expanded persisters (DTEP) were investigated for the mechanisms driving drug tolerance and resistance against Venetoclax. First, we observed that altered expression of apoptotic regulators were associated with Venetoclax resistance in DTEP cells. We indeed observe a significant increase in the anti-apoptotic proteins MCL1 and BCL-XL in DTEP clones, which translated in our observation of improved sensitivity to MCL1 and BCL-xL inhibitors (S63845 and A-1155463 respectively). We performed both whole genome sequencing (WGS) and RNA-seq to evaluate if DTEP cells undergo transcriptional adaptation via genomic or epigenomic regulation and transcriptional reprograming during development of acquired drug resistance. While, WGS analysis didn't show any significant differences between parental and resistant clones, transcriptomic analysis showed both shared and unique transcriptome signatures in the DTEP clones. Gene set enrichment analysis (GSEA) of the common significantly modulated genes in the resistant clones revealed that the genes belonging to the PKA-ERK-CREB pathway were significantly upregulated in resistant clones, while apoptotic genes were downregulated compared to parental cells. Western blot analysis confirmed activation of ERK and the downstream target cAMP response element-binding (CREB) gene in resistant clones; and importantly treatment with the ERK inhibitor U0126 rescued the resistance to Venetoclax, providing a synergistic activity in resistant clones but not in parental cells, with decreased cell viability and increased apoptotic cell death. To evaluate if the ERK pathway was also associated with intrinsic resistance to Venetoclax, we assessed a panel of 24 MM cell lines and then calculated Pearson correlation coefficients between the measured drug activity and individual gene expression levels (by RNA-seq) across all cell lines and subjected the resulting rank-ordered gene list to GSEA. This analysis showed that mechanisms driving the DTEP phenotype are different from those associated with the intrinsic resistance to Venetoclax. RNA processing and splicing pathways were strongly enriched, with high expression of these genes correlating with increased sensitivity. Moreover, among the genes correlated with a resistant phenotype, we observed that the gene G0S2 was significantly downregulated in the resistant cell lines. G0S2 is a tumor suppressor gene that binds and inhibits BCL2. Interestingly, we observed that while G0S2 is downregulated in MM compared to normal plasma cells, t(11:14) patients have a higher expression. We are now in the process of validating G0S2 in MM and its contribution to Venetoclax sensitivity in MM. In conclusion, we here provide evidences of molecular mechanisms of acquired resistance to Venetoclax with activation of the ERK pathway as one of the prime targets. Combining Venetoclax with ERK inhibitor may therefore prevent or overcome the acquired resistance to Venetoclax observed in MM patients. Disclosures Fulciniti: NIH: Research Funding. Munshi:C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy; Adaptive: Consultancy; Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Janssen: Consultancy. Anderson:Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Hematologic Malignancies Exhibit Selective Vulnerability to Inhibition of De Novo Pyrimidine Biosynthesis By AG-636, a Novel Inhibitor of Dihydroorotate Dehydrogenase in Phase 1 Clinical Trials

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1570-1570
Author(s):  
Danielle Ulanet ◽  
Victor Chubukov ◽  
John Coco ◽  
Gabrielle McDonald ◽  
Mya Steadman ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Solid Tumor ◽  
Research Funding ◽  
De Novo ◽  
Phase 1 ◽  
Cancer Cell Lines ◽  
Pyrimidine Biosynthesis ◽  
Employment Equity ◽  
Equity Ownership

Rapidly proliferating cells reprogram metabolism to support increased biosynthetic demands, a feature that can expose targetable vulnerabilities for therapeutic intervention. A chemical biology screen was performed in an effort to identify metabolic vulnerabilities in particular tumor subtypes, and revealed potent and selective activity of a novel dihydroorotate dehydrogenase (DHODH) inhibitor, AG-636, in cancer cell lines of hematologic origin. In contrast, cancer cell lines of solid tumor origin exhibited comparatively poor sensitivity. Evaluation of a lymphoma cell line panel demonstrated broad responsiveness to DHODH inhibition, independent of clinical subtype (e.g. ABC, GCB, double-hit). The on-target cellular activity of AG-636 was evaluated by examining the metabolic effects of AG-636 on cells and by evaluating the ability of extracellular uridine to rescue the effects of AG-636 on proliferation and viability. The metabolic changes incurred upon treatment of cells with AG-636 were consistent with a mechanism of action driven by inhibition of DHODH and de novo pyrimidine biosynthesis. Supraphysiologic concentrations of extracellular uridine rescued the effects of AG-636 on growth and viability as well as the effects on metabolism, further confirming on-target activity. The mechanistic basis for differential sensitivity to AG-636 was assessed by comparing the activity of the de novo pyrimidine biosynthesis and uridine salvage pathways in cancer cell lines of hematologic or solid tumor origin with similar proliferative rates. Differential response to AG-636 could not be attributed to varying abilities to utilize the de novo pyrimidine biosynthesis pathway or to salvage extracellular uridine. Real-time imaging of cells treated with AG-636, along with monitoring of extracellular uridine concentrations, demonstrated immediate effects on the viability of lymphoma cell lines in the setting of depleted extracellular uridine. In contrast, solid tumor cell lines were able to maintain growth for an additional period of time, suggestive of adaptive mechanisms to supply pyrimidine pools and/or to cope with nucleotide stress. The high in vitro activity of AG-636 in cancer cells of hematologic origin translated to xenograft models, including an aggressive, patient-derived xenograft model of triple-hit lymphoma and an ibrutinib-resistant model of mantle cell lymphoma in which complete tumor regression occurred. These studies support the development of AG-636 for the treatment of hematologic malignancies. A phase 1 study has been initiated in patients with relapsed/refractory lymphoma (NCT03834584). Disclosures Ulanet: Agios: Employment, Equity Ownership. Chubukov:Agios: Employment, Equity Ownership. Coco:Agios: Employment, Equity Ownership. McDonald:Agios: Employment, Equity Ownership. Steadman:Agios: Employment, Equity Ownership. Narayanaswamy:Agios: Employment, Equity Ownership. Ronseaux:Agios: Employment, Equity Ownership. Choe:Agios: Employment, Equity Ownership. Truskowski:Agios: Employment, Equity Ownership. Nellore:Aurigene Discovery Technologies: Employment. Rao:Firmus Laboratories: Employment, Equity Ownership. Lenz:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Agios: Research Funding; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy; AstraZeneca: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Employment, Honoraria, Research Funding, Speakers Bureau. Cooper:Agios: Employment, Equity Ownership. Murtie:Agios: Employment. Marks:Agios: Employment, Equity Ownership.

scholarly journals A Novel Spirocyclic Dimer (36-286) Targeting the NF-Kappa B Pathway Displays Potent Anti-Tumor Properties in Chronic Lymphocytic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1186-1186
Author(s):  
Alexandria P Eiken ◽  
Audrey L Smith ◽  
Sarbjit Singh ◽  
Sandeep Rana ◽  
Sunandini Sharma ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Growth ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Protein Translation ◽  
Lymphocytic Leukemia ◽  
Rna Seq ◽  
Resistant Cells

Abstract Introduction: Chronic lymphocytic leukemia (CLL) is an incurable, heterogenetic disease dependent on B cell receptor (BCR) signaling with subsequent nuclear factor-kappa B (NF-κB) activation resulting in the evasion of apoptosis and enhanced malignant B cell growth. Targeted therapies such as ibrutinib (IBR; BTK inhibitor) and venetoclax (VEN; BCL2 antagonist) have revolutionized the management of CLL, however ~20% of patients relapse, signifying the urgent need for novel therapeutics for CLL patients especially those with refractory/relapse (ref/rel) disease. Additionally, various tumor microenvironment (TME) stimuli fuel CLL growth and contribute to drug resistance through the activation of numerous signaling pathways (BCR, CD40R, TLR, BAFFR) and consequential sustained NF-κB activation. Currently, there are no FDA approved drugs that effectively target the NF-κB protein family. Herein we introduce 36-286 (N3), a novel spirocyclic dimer which displays NF-κB inhibitory activity and elicits potent anti-leukemic properties. N3 is a dimer of a spirocyclic α-methylene-γ-butyrolactone analog that covalently binds to surface exposed cysteine residues on NF-κB proteins (IKKβ and P65) (Rana S et al, 2016). Our study aims to investigate N3's mode of action (MOA) and to establish its anti-leukemic effects in CLL including drug-resistant disease, thereby introducing a novel therapeutic option for rel/ref disease. Methods: Cell growth via MTS proliferation assay was determined following treatment with N3 (0.125 - 2 μM) in a panel of malignant B cell lines [CLL (HG3, MEC1, OSUCLL), diffuse large B cell lymphoma (Pfeiffer, RC, RIVA), mantle cell lymphoma (Jeko1)], and in patient derived CLL cells stimulated with CpG ODN 2006 (CpG; 3.2 μM). Viability testing of normal B cells isolated from healthy donors was conducted following N3 treatment. Anti-tumor properties of N3 (1 - 2 μM; 4h) in the HG3 and OSUCLL cell lines were further confirmed under conditions mimicking different TME stimuli such as α-IgM (10 μg/mL), CD40L (100 ng/mL), BAFF (50 ng/mL) or CpG (3.2 μM). Protein expression of oncogenic MYC, select NF-κB pathway proteins (IKKα, IKKβ, P65, IκBα, RelB) and the anti-apoptotic protein MCL1 was determined following treatment with N3 (0.25 - 2 μM; 4h) by immunoblot (IB). Next, we induced IBR resistance in HG3 cells by prolonged exposure to increasing IBR concentrations (~10-15 fold its IC 50 in parental cells). Cell proliferation via MTS was determined following treatment with N3 on these resistant cells. To gain insight on the potential MOA of N3 in CLL, we adapted a proteomics-based approach (TMT labeled mass spectrometry) and conducted RNA-seq in OSUCLL cells treated with N3 (1 - 2 μM) for up to 24 h. Subsequent pathway analysis was performed to identify the top factors modulated by N3. Results: N3 showed remarkable efficacy (IC 50 < 0.6 μM) across all the malignant B cell lines evaluated while sparing normal B cells. In CpG stimulated primary CLL, N3 resulted in marked anti-leukemic effects (0.125 μM) comparable to IBR (1 μM). N3 induced cell apoptosis in CLL cell lines in a dose-dependent manner with marked PARP cleavage. Furthermore, our IB analyses of N3 treated CLL cell lines showed reduced levels of NF-κB pathway proteins, MYC and MCL1. Notably, N3 was effective in reducing levels of the above-mentioned proteins in the presence of the various TME stimuli. Strikingly, N3 maintained its cytotoxic effects in ibrutinib resistant HG3 cells. Studies to confirm N3's cytotoxicity in VEN resistant CLL cells are ongoing. Top ten pathways from both proteomics and RNA-seq analyses revealed an upregulation of the unfolded protein response (UPR) and inhibition of cap-dependent protein translation. IB analyses of select factors related to UPR (CHOP, XBP1, PERK, IRE1) and protein translation (eIF2α, 4E-BP1, PDCD4) in N3 treated CLL cells validated our omics' findings. Efforts to identify the proteome wide direct targets of N3 in CLL cells are currently underway. Conclusion: N3 is a novel pre-therapeutic lead that targets multiple survival and proliferation pathways through the inhibition of NF-κB activity and upregulation of UPR. We show that its highly cytotoxic in tumor B cells while sparing normal B cells. Moreover, N3 sustained its anti-tumor properties under different TME stimuli and in IBR resistant cells, indicating the potential use of this compound in rel/ref patients following evaluation in murine CLL models. Disclosures No relevant conflicts of interest to declare.

Abstract B24: De novo and acquired resistance to first-line standard therapy in colorectal cancer: from cell lines to metastatic tumors

2012 ◽  
Vol 18 (10 Supplement) ◽  
pp. B24-B24
Author(s):  
Raquel Aloyz ◽  
Caroline Rousseau ◽  
Zuanel Diaz ◽  
Adriana Aguilar-Mahecha ◽  
Mark Basik ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Standard Therapy ◽  
De Novo ◽  
Acquired Resistance ◽  
First Line ◽  
Metastatic Tumors ◽  
Line Standard

The IKK Inhibitor PS1145 Allows to Overcome Imatinib Resistance.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2940-2940
Author(s):  
Daniela Cilloni ◽  
Francesca Messa ◽  
Francesca Arruga ◽  
Ilaria Defilippi ◽  
Alessandro Morotti ◽  
...  
Keyword(s):  
Cell Lines ◽  
Phase 1 ◽  
Chronic Phase ◽  
Colony Growth ◽  
Resistant Cell ◽  
Proliferation Rate ◽  
Imatinib Resistance ◽  
Inhibition Of Proliferation ◽  
Imatinib Resistant ◽  
Resistant Cells

Abstract Since a number of CML patients are resistant to Imatinib, additional molecular defects should be identified and targeted to improve the therapeutic strategies. A constitutive NF-kB activity has been demonstrated in several hematologic malignancies, therefore NF-kB blocking approaches have been introduced as antineoplastic strategies. The phosphorilation of IKB by IKK leads to IKB degradation by the proteasome, so freeing NF-kB to enter the nucleus and activate transcription. IKK may therefore represent an attractive target for molecular therapies. The aim of the study was to evaluate the effects of the IKK inhibitor PS1145 (Millenium) in CML cell lines and patients sensitive and resistant to Imatinib. K562 and KCl cells both sensitive (s) and resistant (r) to Imatinib and the BM cells collected from 13 CML patients were incubated with PS1145 10 μM, with Imatinib 1 μM and with the combination of the two drugs for 24 and 48 hrs. 11 out of 13 patients were in chronic phase, 1 in accelerate phase and 1 in blastic phase. 3 out of 11 were cytogenetic resistant, one was also hematologically resistant. The NF-kB activity was evaluated by ELISA method, the proliferation rate by MTT assay, apoptosis by FACS. Immunofluorescence was use to identify the NF-kB localization. Moreover colony growth was evaluated in control and treated samples. PS1145 was able to reduce the DNA binding activity of NF-kB of 90% and 85% respect to the control in K562 and KCL cells.This was confirmed by the prevailing cytoplasmatic localization of NF-kB after PS1145 incubation. In K562s and KCls treated with Imatinib, we observed a reduction of proliferation of 48% and 37% respectively. By contrast no effect on proliferation was observed in K562r and KClr. The incubation with PS1145 inhibited the proliferation of 38% and 15% in K562s and KCls, and of 22% and 28% in K562r and KClr. Interestingly in resistant cell lines the association of Imatinib and PS1145 induced a marked inhibition of proliferation of 87% in K562r and 82% in KClr. Similarly, PS1145 inhibited BM cells proliferation of 30% (range 11% to 65%). Imatinib incubation of BM cells from sensitive patients reduced the proliferation rate of 41% (range 29%–55%) but no effects were observed in resistant patients. In the three resistant patients the incubation with both compounds resulted in an increased block of proliferation respect to PS1145 alone with an inhibition of 42%, 49% and 58% after 24 hrs and 70%, 77% and 79% after 48 hrs. Imatinib plus PS1145 induced a significant increase of apoptosis from 7% to 69% in K562r and from 9 % to 71% in KClr. In agreement this association induced 48%, 52% and 39% of apoptotic cells in BM from the three resistant patients and a colony growth inhibition of 86%. Our data clearly demonstrated that the IKK inhibitor PS1145 is able to induce growth arrest and apoptosis in CML cell lines and BM cells. This effect is more sound in Imatinib resistant cells treated with the association of Imatinib and PS1145. Although the exact mechanism of action of PS1145 in resistant cells is at present a pure speculation, these data may suggest an intriguing mechanism to induce apoptosis in imatinib resistant cells based on imatinib resistance itself. The combination of Imatinib and the IKK inhibitor could therefore represent a valid approach for the treatment of CML patients resistant to Imatinib therapy.

Mechanisms of Acquired Resistance to Venetoclax in Preclinical AML Models

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 328-328 ◽  
Author(s):  
Qi Zhang ◽  
Rongqing Pan ◽  
Lina Han ◽  
Ce Shi ◽  
Stephen E. Kurtz ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Synergistic Effects ◽  
Acquired Resistance ◽  
Parental Cell ◽  
Resistant Cell ◽  
Flt3 Inhibitors ◽  
Resistant Cells

Abstract BH3-mimetic ABT-199 (venetoclax, VEN) is a selective small-molecule antagonist of the anti-apoptotic BCL-2 protein. It binds to BCL-2 specifically, causing the release of pro-apoptotic BAX and BH3-only proteins and induction of cell death. Our studies indicated that AML is a BCL-2 dependent disease that, in pre-clinical studies, responds robustly to VEN by induction of apoptotic cell death (Pan et al., Cancer Discovery 2014). As a single agent, VEN demonstrated clinical activity in relapsed/refractory AML, yet patients who initially responded ultimately developed resistance and progressed. In this study we investigated mechanisms of acquired resistance to VEN in preclinical AML models. First, we generated 5 VEN-resistant cell lines (OCI-AML2, Kasumi, KG-1, MV4;11 and Molm13; with VEN cell-killing IC50s of 0.021µM, 0.046µM, 0.073µM, 0.020µM and 0.050µM, respectively) by exposing the cells to gradually increasing VEN concentrations. The IC50s of resistant cells are 15.2µM, 5.7µM, 31.6µM, 11.4µM and 15.4µM (124-723-fold greater than their parental counterparts). Protein analysis of resistant cells using immunoblotting demonstrated increased expression of MCL-1, a known resistance factor to VEN, in 4 resistant cell lines (OCI-AML2, KG-1, Mv4;11 and Molm13); and BCL-XL increase in MV4;11 and Molm13 resistant cells. To characterize the functional role of MCL-1 and BCL-XL in resistance to VEN, we co-treated parental and resistant cells with novel MCL-1 and BCL-XL- selective inhibitors (A-1210477 and A-1155463). The combination of VEN with A-1210477 or A-1155463 showed synergistic growth inhibition in all 5 parental cell lines (combination indices (CI) for A-1210477 were 0.15-0.62; CI for A-1155463 were 0.33-0.51, except >3 for KG-1). Notably, 4 out of 5 resistant cell lines (OCI-AML2, Kasumi, MV4;11, Molm13) became more sensitive to MCL-1 selective inhibitor A-1155463 but not to BCL-XL inhibitor A-1210477. However, no further effects were seen in resistant cells when combined with VEN. We next compared sensitivity of three paired parental and resistant cell lines (OCI-AML2, MV4;11 and Molm13) to a library of 130 specific small-molecule inhibitors (Tyner, et.al.. Cancer Res. 2013). Cells were co-treated with VEN and each specific inhibitor, and drug target scores were calculated based on the IC50 of measured effectiveness of panel drugs against the cells. The screening revealed modulation of sensitivity to mTOR, MEK, and FLT3 pathways in resistant cells (Fig.1C). To confirm these findings, we next co-treated AML cells with VEN and specific inhibitors of the mTOR pathway (rapamycin and AZD2014) or MEK pathway (CI1040) in all 5 paired parental and resistant cell lines; or with FLT3 inhibitors (quizartinib and sorafenib) in parental and resistant MV4;11 and Molm13, which harbor FLT3-ITD. The combination of VEN and AZD2014 achieved synergistic effects in all 5 parental cell lines (CI AZD2014: 0.08-0.94), and VEN/rapamycin were synergistic in 3 parental cell lines (CI rapamycin: 0.00-0.55, except 1.76 for KG-1 and 1.59 for Molm13). Combination of VEN with CI1040 achieved synergy in OCI-AML2, Kasumi, MV4;11 and Molm13 parental cell lines (CI: 0.14-0.61). Finally, VEN/FLT3 inhibitors achieved synergistic effects in MV4;11 and Molm13 parental cell lines (CI quizartinib: 0.66-0.69; CI sorafenib: 0.64-0.71). The resistant cell lines exhibited sensitivity to these inhibitors as single agents, and no synergistic effects were seen when combined with VEN. We have further induced in vivo resistance in two primary AML xenografts by treating NSG mice engrafted with 2nd passage AML cells with 100 mg/kg Q.D. VEN for 4 weeks followed by harvest of leukemic cells that repopulated the mouse after treatment discontinuation. While the proteomics, gene expression (RNAseq) and drug screening assays are in progress, preliminary immunoblotting studies demonstrated decreased expression of BCL-XL and BCL-2 (Fig.1B). In summary, we identified multiple mechanisms of acquired resistance to VEN, which ultimately modulate the balance between pro- and anti-apoptotic BCL-2 family members. Our studies indicate that upfront combination of VEN with selective inhibitors of MCL-1, or with inhibitors of specific signaling pathways, can synergistically induce apoptosis in AML cells and conceivably prevent emergence of VEN resistance. Disclosures Leverson: AbbVie: Employment, Equity Ownership. Tyner:Aptose Biosciences: Research Funding; Constellation Pharmaceuticals: Research Funding; Janssen Pharmaceuticals: Research Funding; Array Biopharma: Research Funding; Incyte: Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

scholarly journals Chromatin and RNA Maps Reveal Regulatory Long Noncoding RNAs in Mouse

2015 ◽  
Vol 36 (5) ◽  
pp. 809-819 ◽  
Author(s):  
Gireesh K. Bogu ◽  
Pedro Vizán ◽  
Lawrence W. Stanton ◽  
Miguel Beato ◽  
Luciano Di Croce ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
De Novo ◽  
Noncoding Rnas ◽  
Mouse Cell ◽  
Long Noncoding Rnas ◽  
Chromatin State ◽  
Rna Seq ◽  
Gene Encoding ◽  
Protein Coding

Discovering and classifying long noncoding RNAs (lncRNAs) across all mammalian tissues and cell lines remains a major challenge. Previously, mouse lncRNAs were identified using transcriptome sequencing (RNA-seq) data from a limited number of tissues or cell lines. Additionally, associating a few hundred lncRNA promoters with chromatin states in a single mouse cell line has identified two classes of chromatin-associated lncRNA. However, the discovery and classification of lncRNAs is still pending in many other tissues in mouse. To address this, we built a comprehensive catalog of lncRNAs by combining known lncRNAs with high-confidence novel lncRNAs identified by mapping andde novoassembling billions of RNA-seq reads from eight tissues and a primary cell line in mouse. Next, we integrated this catalog of lncRNAs with multiple genome-wide chromatin state maps and found two different classes of chromatin state-associated lncRNAs, including promoter-associated (plncRNAs) and enhancer-associated (elncRNAs) lncRNAs, across various tissues. Experimental knockdown of an elncRNA resulted in the downregulation of the neighboring protein-codingKdm8gene, encoding a histone demethylase. Our findings provide 2,803 novel lncRNAs and a comprehensive catalog of chromatin-associated lncRNAs across different tissues in mouse.

scholarly journals Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Kana Oiwa ◽  
Naoko Hosono ◽  
Rie Nishi ◽  
Luigi Scotto ◽  
Owen A. O’Connor ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Acquired Resistance ◽  
3D Culture ◽  
Resistance Mechanisms ◽  
Resistant Cell ◽  
Gene Set Enrichment Analysis ◽  
Synergistic Activity ◽  
Mechanisms Of Resistance ◽  
Resistant Cells

Abstract Background Pralatrexate (PDX) is a novel antifolate approved for the treatment of patients with relapsed/refractory peripheral T-cell lymphoma, but some patients exhibit intrinsic resistance or develop acquired resistance. Here, we evaluated the mechanisms underlying acquired resistance to PDX and explored potential therapeutic strategies to overcome PDX resistance. Methods To investigate PDX resistance, we established two PDX-resistant T-lymphoblastic leukemia cell lines (CEM and MOLT4) through continuous exposure to increasing doses of PDX. The resistance mechanisms were evaluated by measuring PDX uptake, apoptosis induction and folate metabolism-related protein expression. We also applied gene expression analysis and methylation profiling to identify the mechanisms of resistance. We then explored rational drug combinations using a spheroid (3D)-culture assay. Results Compared with their parental cells, PDX-resistant cells exhibited a 30-fold increase in half-maximal inhibitory concentration values. Induction of apoptosis by PDX was significantly decreased in both PDX-resistant cell lines. Intracellular uptake of [14C]-PDX decreased in PDX-resistant CEM cells but not in PDX-resistant MOLT4 cells. There was no significant change in expression of dihydrofolate reductase (DHFR) or folylpolyglutamate synthetase (FPGS). Gene expression array analysis revealed that DNA-methyltransferase 3β (DNMT3B) expression was significantly elevated in both cell lines. Gene set enrichment analysis revealed that adipogenesis and mTORC1 signaling pathways were commonly upregulated in both resistant cell lines. Moreover, CpG island hypermethylation was observed in both PDX resistant cells lines. In the 3D-culture assay, decitabine (DAC) plus PDX showed synergistic effects in PDX-resistant cell lines compared with parental lines. Conclusions The resistance mechanisms of PDX were associated with reduced cellular uptake of PDX and/or overexpression of DNMT3B. Epigenetic alterations were also considered to play a role in the resistance mechanism. The combination of DAC and PDX exhibited synergistic activity, and thus, this approach might improve the clinical efficacy of PDX.

scholarly journals Anti-Leukemia Effect of FF-10501-01, a Novel Inosine 5'-Monophosphate Dehydrogenase Inhibitor, in Advanced Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS), Including Hypomethylating Agent (HMA) Failures

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3800-3800 ◽  
Author(s):  
Guillermo Garcia-Manero ◽  
Hui Yang ◽  
Zhihong Fang ◽  
Courtney DiNardo ◽  
Elias Jabbour ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
De Novo ◽  
Phase 1 ◽  
Leukemia Cell ◽  
Clinical Activity ◽  
Guanine Nucleotide ◽  
Leukemia Cell Lines ◽  
Induced Apoptosis

Abstract Inosine 5'- monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes de novo synthesis of the guanine nucleotide and is overexpressed in both hematologic and solid tumors. FF-10501-01 is a potent new competitive IMPDH inhibitor. We investigated the anti-leukemia effect of FF-10501-01 in AML cell lines and in a Phase 1 clinical study in advanced AML and MDS, including HMA failures. Thirteen leukemia cell lines were studied, including 5 parental AML cell lines and their HMA-resistant derivatives (MOLM13, SKM1, HL60, TF1, and U937), and 3 other AML cell lines (KG1, HEL, and OCI-AML3). Cell proliferation was determined using trypan blue analysis. Flow cytometry was performed to detect drug-induced apoptosis and cell cycle analysis. High-performance liquid chromatography (HPLC) was performed to detect the intracellular concentrations of guanine nucleotides. Mycophenolic acid-treated cells were used as positive control. Effect of guanosine supplement on FF-10501-01 treatment was evaluated. Within 72 hours of treatment, FF-10501-01 inhibited proliferation of all 13 AML cell lines. The IC50 of FF-10501-01 ranged between 4.3 and 144.5 µM. MOLM13 was the most sensitive leukemia cell line, whereas the decitabine-resistant TF1 cell line was the most resistant. FF-10501-01-induced apoptosis was observed in all cell lines. Increased numbers of cells in G1 phase and decreased numbers in S phase were observed in MOLM13, SKM1 and TF1 cell lines treated with <100 µM FF-10501-01. Decreased intracellular concentrations of guanine nucleotides were observed in MOLM13 and SKM1 cell lines treated with 3 to 30 µM of FF-10501-01 for 24 hours. Proliferation was partially rescued after 72 hours of treatment with 3 µM guanosine and FF-10501-01 in MOLM-13, HL60 cells and their HMA-resistant derivatives. No treatment synergy was observed with the combination of FF-10501-01 with HMAs in MOLM-14 and HL-60 or their HMA-resistant cell lines. In summary, FF-10501-01 produced potent anti-proliferative and apoptotic effects on AML cell lines through inhibition of de novo guanine nucleotide synthesis. In view of these pre-clinical findings, we performed a standard 3+3 dose-escalation Phase 1 trial to access the safety and clinical activity of FF-10501-01 in patients with advanced AML, MDS and chronic myelomonocytic leukemia (CMML). Eligibility criteria: age ³ 18 years, high risk MDS/CMML, AML with documented PD following previous therapy, AML ≥ 60 years of age and not a candidate for other therapy, adequate renal and hepatic function, and no known history of significant cardiac disease. Sixteen patients (12 AML, 4 MDS) have been enrolled in 5 dose cohorts (50 - 400 mg/m2 PO BID) for 14 days on/14 days off each 28-day cycle, including 8 M and 8 F. Median (range) values: age 75.3 yrs (59.1 - 88.6); bone marrow blasts for AML patients 40.5% (12 - 71), for MDS patients 10% (6 - 13), or 30% overall (6 - 71); and prior treatment regimens 2.5 (1 - 6). All patients relapsed from, or progressed on, prior HMAs. Mutations in FLT3, NPM1, GATA2, TET2, ASXL1, DNMT3A and/or MDM2 were present in 4/16 (25%) patients. The median number of FF-10501-01 cycles received to date is 1.5 (range 1 - 10). No DLTs or drug-related serious adverse events (AEs) have been observed and FF-10501-01 has been very well tolerated through 5 - 10 cycles. The most frequent drug-related AEs have been Gr 1-2 nausea, diarrhea and fatigue. Drug-related Gr 4 prolonged thrombocytopenia and Gr 4 prolonged neutropenia were reported in one patient at 200 mg/m2 BID. Two partial responses (PRs) have been achieved in 1 patient each at 50 and 100 mg/m2 BID after 3 cycles, 7 (50%) patients demonstrated long-term stable disease over 2 - 10 cycles, and 4 patients have remained on study drug through 5 - 10 cycles and are still ongoing. Updated safety and efficacy data, including PK/PD, will be presented at the meeting. FF-10501-01 is a promising new agent for the treatment of advanced AML and MDS. Preclinical activity was seen in multiple leukemia cell lines. In a Phase 1 trial, clinical activity with PRs, prolonged disease stabilization and a highly tolerable safety profile were observed. The Phase 2 expansion phase will be initiated soon. Disclosures DiNardo: Novartis: Research Funding. Pemmaraju:Stemline: Research Funding; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria. Smith:Westat Corporation: Employment. Iwamura:FUJIFILM Corporation: Employment. Gipson:Strategia Therapeutics, Inc.: Employment. Rosner:Strategia Therapeutic, Inc.: Employment. Madden:Strategia Therapeutics, Inc.: Employment. Myers:Strategia Therapeutics, Inc.: Employment. Paradiso:Strategia Therapeutics, Inc.: Employment.

scholarly journals AR mRNA stability is increased with AR-antagonist resistance via 3′UTR variants

2020 ◽  
Vol 9 (1) ◽  
pp. 9-19 ◽  
Author(s):  
D A Dart ◽  
K Ashelford ◽  
W G Jiang
Keyword(s):  
Prostate Cancer ◽  
Cell Lines ◽  
Mrna Stability ◽  
Advanced Prostate Cancer ◽  
Actinomycin D ◽  
Acquired Resistance ◽  
Resistant Cell ◽  
Rna Seq ◽  
Serum Samples ◽  
Mrna Variant

Advanced prostate cancer is often treated with AR antagonists which target the androgen receptor (AR) on which the growth of the tumour depends. Prostate cancer often develops AR-antagonist resistance via a plethora of mechanisms, many of which are as yet unknown, but it is thought that AR upregulation or AR ligand-binding site mutations, may be responsible. Here we describe the production of cell lines based on LNCaP and VCaP, with acquired resistance to the clinically relevant AR antagonists, bicalutamide and enzalutamide. In these resistant cells, we observed, via RNA-seq, that new variants in the 3′UTR of the AR mRNA were detectable and that the levels were increased both with AR-antagonist treatment and with hormonal starvation. Around 20% of AR transcripts showed a 3 kb deletion within the 6.7 kb 3′UTR sequence. Actinomycin D and luciferase fusion studies indicated that this shorter mRNA variant was inherently more stable in anti-androgen-resistant cell lines. Of additional interest was that the AR UTR variant could be detected in the sera of prostate cancer patients in a cohort of serum samples collected from patients of Gleason grades 6–10, with an increasing level correlated to increasing grade. We hypothesise that the shorter AR UTR variant is a survival adaptation to low hormone levels and/or AR-antagonist treatment in these cells, where a more stable mRNA may allow higher levels of AR expression under these conditions.

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