Jorge DiMartino

The DOT1L inhibitor pinometostat reduces H3K79 methylation and has modest clinical activity in adult acute leukemia

Pinometostat (EPZ-5676) is a first-in-class small-molecule inhibitor of the histone methyltransferase disrupter of telomeric silencing 1-like (DOT1L). In this phase 1 study, pinometostat was evaluated for safety and efficacy in adult patients with advanced acute leukemias, particularly those involving mixed lineage leukemia (MLL) gene rearrangements (MLL-r) resulting from 11q23 translocations. Fifty-one patients were enrolled into 6 dose-escalation cohorts (n = 26) and 2 expansion cohorts (n = 25) at pinometostat doses of 54 and 90 mg/m2 per day by continuous intravenous infusion in 28-day cycles. Because a maximum tolerated dose was not established in the dose-escalation phase, the expansion doses were selected based on safety and clinical response data combined with pharmacodynamic evidence of reduction in H3K79 methylation during dose escalation. Across all dose levels, plasma pinometostat concentrations increased in an approximately dose-proportional fashion, reaching an apparent steady-state by 4-8 hours after infusion, and rapidly decreased following treatment cessation. The most common adverse events, of any cause, were fatigue (39%), nausea (39%), constipation (35%), and febrile neutropenia (35%). Overall, 2 patients, both with t(11;19), experienced complete remission at 54 mg/m2 per day by continuous intravenous infusion, demonstrating proof of concept for delivering clinically meaningful responses through targeting DOT1L using the single agent pinometostat in MLL-r leukemia patients. Administration of pinometostat was generally safe, with the maximum tolerated dose not being reached, although efficacy as a single agent was modest. This study demonstrates the therapeutic potential for targeting DOT1L in MLL-r leukemia and lays the groundwork for future combination approaches in this patient population. This clinical trial is registered at www.clinicaltrials.gov as NCT01684150.

Abstract 2280: Functional characterization of combining epigenetic modifiers azacitidine and AG-221 in the TF-1:IDH2R140Q AML model

Approximately 15% of AML patients have an IDH2 mutation which leads to production of the oncometabolite 2-hydroxyglutarate (2HG). Accumulation of 2HG inhibits aKG-dependent DNA and histone demethylases, resulting in epigenetic disregulation, which in turn leads to a block in cellular differentiation, promoting AML. AG-221 is a selective inhibitor of IDH2 mutant enzyme and is in development for AML patients carrying IDH2 mutation. Clinical responses to AG-221 therapy have been observed, including decreases in blast percentages and evidence of differentiated functional blood cells; however, preliminary analysis indicated that the mutant allelic burden was not reduced in a majority of subjects treated with AG-221. Azacitidine (AZA) is another epigenetic modifying agent with clinical activity in AML. We hypothesized that combining AG-221 with AZA could synergize in releasing the differentiation block in IDH2-mutant AML cells and enhance cell killing. TF-1:IDH2R140Q are human erythroleukemia cells engineered to express R140Q-mutant IDH2 and model the differentiation block conferred by 2HG accumulation. TF-1:IDH2R140Q cells were treated with AG-221, AZA or the combination of AG-221 + AZA, and measures of cell differentiation (hemoglobinization, KLF1 and HBG qRT-PCR, CD34/CD38 flow cytometry) and death (IncuCyte Zoom caspase 3/7) were evaluated. Measures of cell differentiation were evaluated in TF-1:IDH2R140Q cells, using an in vitro erythropoietin differentiation assay. Single agent AG-221 and AZA increased heme production in a dose-dependent manner, as evidenced by increased red color of cells. With AZA + AG-221 combination, hemoglobinization was greater than with single agents. Dose-dependent increases in RNA expression of differentiation markers KLF1 and HBG were observed with single agents, and the combination resulted in additive, or greater than additive, increases. Quantification of hematopoietic stem /progenitor cell populations demonstrated that as single agents, both AG-221 and AZA reduced CD34+/CD38+ and CD34+/CD38- cell populations, and the combination resulted in additive or greater than additive decreases. Real-time quantification of cell death showed that single agent AG-221 had no effect, while single agent AZA increased apoptosis. Concurrent combination of AZA + AG-221 increased cell death beyond that of single agents. Whole genome expression data (RNA-seq) showed enrichment of differentiation and cell death gene signatures with the combination when compared to single agents alone. We have demonstrated the beneficial effects of combining AG-221 + AZA in the TF-1:IDH2R140Q AML cell line, including greater than additive increases in hemoglobinization and expression of differentiation markers, reduced stem /progenitor cell populations, and potentiation of death. Further exploration of the molecular mechanism of the combination is ongoing. Citation Format: Vivek S. Chopra, Brian Avanzino, Konstantinos Mavrommatis, Adam Olshen, Jorge DiMartino, Kyle J. MacBeth. Functional characterization of combining epigenetic modifiers azacitidine and AG-221 in the TF-1:IDH2R140Q AML model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2280.

Abstract A120: A Phase Ib study of CC-486 (Oral Azacitidine) as a priming agent for carboplatin or NAB-paclitaxel in subjects with relapsed and refractory solid tumors.

Background: CC-486, an oral formulation of 5-azacitidine, induces global genomic hypomethylation and clinical responses in MDS patients at doses of 200 mg and 300 mg/day for 14 or 21/28 days with acceptable safety. This non-randomized, multi-center Phase Ib study was designed to determine whether CC-486 can be delivered safely as priming for carboplatin or NAB-Paclitaxel (ABI-007) in patients with solid tumors. Methods: In Part 1 of this study, patients with refractory solid tumors were assigned to Arm A: CC-486 days 1 - 14 with carboplatin AUC 4 on day 8 of a 21 day cycle (n = 13) or Arm B: CC-486 days 1 - 14 with ABI-007 100 mg/m2 given weekly starting on Day 8 of Cycle 1 (n = 24). 200 mg and 300 mg dose levels (DLs) of CC-486 were evaluated in cohorts of 6 to identify a recommended Part 2 dose (RP2D). Results: On Arm A, 200 mg and 300 mg doses of CC-486 were tolerated. There was one DLT (Grade 3 pericardial effusion) at the 300 mg DL. Other related treatment-emergent adverse events (TEAEs) included anemia (53.8%) and neutropenia (46.3%). On Arm B, there were 2 DLTs of neutropenia at the 200 mg DL. The protocol was amended to give ABI-007 on days 8 and 15/21. No DLTs were encountered on 200 mg of CC-486 with intermittent ABI-007. At 300 mg, the MTD was exceeded with 2 DLTs of neutropenia (cholangiocarcinoma, pancreatic). Other common TEAEs in Arm B included nausea/vomiting (45.8%) and peripheral neuropathy (50%). Evidence of activity seen at both DLs included hypomethylation in PBMCs, 3 PRs in Arm B and 5 subjects with stable disease (> 4 mos) in Arm A. CC-486 plasma exposure showed high interpatient variability but was higher at 300 mg than 200 mg. Incidence and severity of AEs did not differ significantly between the 2 DLs. 300 mg was selected as the RP2D in combination with carboplatin and 200 mg in combination with ABI-007. Conclusion: CC-486 dosed 14/21 days is tolerated as a priming agent with carboplatin and ABI-007. Both combinations show evidence of clinical activity. Expansion cohorts at the RP2D of Arm A (relapsed/refractory bladder or ovarian carcinoma) and Arm B (relapsed/refractory NSCLC or pancreatic carcinoma) are enrolling to further characterize safety, PK/PD and anti-tumor activity. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A120. Citation Format: Patricia LoRusso, Drew Rasco, Johanna Bendell, Jasgit Sachdev, Ramesh Ramanathan, Glenn Weiss, Pamela Munster, William J. Edenfield, Kejian Liu, Anne Blackwood-Chirchir, Jorge DiMartino, Jorge DiMartino, Daniel D. Von Hoff. A Phase Ib study of CC-486 (Oral Azacitidine) as a priming agent for carboplatin or NAB-paclitaxel in subjects with relapsed and refractory solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A120.

Multi-omics analysis of AML cells treated with azacitidine reveals highly variable cell surface proteome remodeling

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are diseases of abnormal hematopoietic differentiation with aberrant epigenetic alterations. Azacitidine (AZA) is a DNA methyltransferase inhibitor (DNMTi) widely used to treat MDS and AML, yet the impact of AZA on the cell surface proteome has not been defined. To identify potential therapeutic targets for use in combination with AZA in AML patients, we investigated the effects of AZA treatment on four AML cell lines (KG1a, HL60, HNT34, and AML193), representing different stages of differentiation. The effect of AZA treatment on these cell lines was characterized at three levels: the DNA methylome (methylation array), the transcriptome (gene expression array), and the cell surface proteome (glycoprotein capture with SILAC labeling). Untreated AML cell lines showed substantial overlap in their methylomes, transcriptomes, and cell surface proteomes. AZA treatment globally reduced DNA methylation in all cell lines, but changes in the transcriptome and surface proteome were subtle and differed among the cell lines. Transcriptome analysis identified five commonly up-regulated coding genes upon AZA treatment in all four cell lines, TRPM4 being the only gene encoding a surface protein, and surface proteomics analysis found no commonly regulated proteins. Gene Set Enrichment Analysis (GSEA) of differentially-regulated RNA and surface proteins showed a decrease in metabolism pathways and an increase in immune defense response pathways. As such, AZA treatment in four AML cell lines had diverse effects at the individual gene and protein level, but converged to regulation of metabolism and immune response at the pathway level. Given the heterogeneous response of AZA in the four cell lines at the gene and protein level, we discuss potential therapeutic strategies for combinations with AZA.

Phase I Study of CC-486 Alone and in Combination With Carboplatin or nab‑paclitaxel in Patients With Relapsed or Refractory Solid Tumors

Purpose: This large two-part, three-arm phase I study examined the safety and tolerability of CC-486 (an oral formulation of azacitidine, a hypomethylating agent) alone or in combination with the cytotoxic agents, carboplatin or nab-paclitaxel, in patients with advanced unresectable solid tumors. Patients and Methods: Part 1 (n = 57) was a dose escalation of CC-486 alone (arm C) or with carboplatin (arm A) or nab-paclitaxel (arm B). The primary endpoint was safety, MTD, and recommended part 2 dose (RP2D) of CC-486. In part 2 (n = 112), the primary endpoint was the safety and tolerability of CC-486 administered at the RP2D for each treatment arm, in tumor-specific expansion cohorts. Secondary endpoints included pharmacokinetics, pharmacodynamics, and antitumor activity of CC-486. Results: At pharmacologically active doses CC-486 in combination with carboplatin or nab-paclitaxel had a tolerable safety profile and no drug–drug interactions. The CC-486 RP2D was determined as 300 mg (every day, days 1–14/21) in combination with carboplatin (arm A) or as monotherapy (arm C); and 200 mg in the same dosing regimen in combination with nab-paclitaxel (arm B). Albeit limited by the small sample size, CC-486 monotherapy resulted in partial responses (three/eight) and stable disease (four/eight) in patients with nasopharyngeal cancer. Three of the stable disease responses lasted more than 150 days. Conclusions: CC-486 is well tolerated alone or in combination with carboplatin or nab-paclitaxel. Exploratory analyses suggest clinical activity of CC-486 monotherapy in nasopharyngeal cancer and provided the basis for an ongoing phase II clinical trial (ClinicalTrials.gov identifier: NCT02269943). Clin Cancer Res; 24(17); 4072–80. ©2018 AACR.

Abstract B82: Pinometostat (EPZ-5676) enhances the antiproliferative activity of MAP kinase pathway inhibitors in MLL-rearranged leukemia cell lines

Despite recent advances in identifying treatment options for acute leukemias, those bearing rearrangements of MLL (MLL‑r) remain a population with significant unmet medical need. Phase 1 clinical trials are underway to investigate the single agent activity of pinometostat (EPZ‑5676), a first‑in‑class inhibitor of the histone methyltransferase DOT1L, for the treatment of patients withMLL‑r acute leukemias. Preliminary results of the adult Phase 1 clinical trial indicate evidence of clinical activity including complete responses in a subset of patients (Blood Dec 2014, 124 (21) 387). While this activity is encouraging, given that the backbone of effective leukemia therapies are combination regimens, investigations into potential pinometostat combinations are of interest. We have previously shown in vitro synergistic anti‑proliferative activity when pinometostat was combined with the current standard of care drugs for acute leukemias, cytarabine and daunorubicin (Klaus, Iwanowicz et al., 2014); the greatest combination benefits with these drugs were observed in cell lines with nanomolar pinometostat IC50 values. Here we report results of an expanded effort, across a leukemia cell line panel to identify novel pinometostat combinations. A high‑throughput screening platform consisting of a library of approved oncology drugs, emerging therapies and tool compounds (n = 250), was utilized to identify synergistic anti‑proliferative activity with pinometostat against a panel of cell lines with a range of reported sensitivities to pinometostat alone (IC50: 0.075 to >3 μM). The study paradigm for this effort consisted of pretreating each cell line for 7 days (MLL‑r: OCI‑AML‑4, ML2, THP‑1, RS4‑11, MOLM‑13, and non‑MLL‑r SKM‑1) with pinometostat prior to the addition of the enhancer agent for 3 days. Combination benefit was calculated by measuring the growth inhibition (GI), and the GI100 potency change of each enhancer compound in the presence of pinometostat or vehicle control. Of the 250 compounds, 12 target classes were observed to have a GI100 shift ≥ 2-fold in at least two cell lines. Among the most compelling findings was synergistic activity of pinometostat with several modulators of the MAP kinase pathway (e.g., trametinib, an approved MEK inhibitor) in multiple MLL‑r cell lines. Study of dosing schedule of the combination of pinometostat with trametinib revealed that all schedules, no matter the order of compound addition, demonstrated combination benefit. Pretreatment with the DOT1L inhibitor, however, elicited dramatic cell killing at physiologically achievable concentrations. When compared to monotherapy, our results indicate that combinatorial treatment of pinometostat with trametinib boosts the inhibitory effect on cell lines sensitive and resistant to DOT1L inhibition. Taken together, these findings suggest that suppression of DOT1L activity prior to MEK inhibition may have advantages over monotherapy of either agent. Additional preclinical investigations into the mechanistic basis of the observed synergy between trametinib and pinometostat are ongoing in support of future potential clinical trials. Citation Format: Alejandra Raimondi, Christine R. Klaus, Jeffrey A. Keats, Scott R. Daigle, Robert A. Copeland, Jorge DiMartino, Jesse J. Smith, Stephen J. Blakemore. Pinometostat (EPZ-5676) enhances the antiproliferative activity of MAP kinase pathway inhibitors in MLL-rearranged leukemia cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B82.

Abstract 5383: DOT1L inhibitor EPZ-5676 synergizes with cytarabine and azacitidine in preclinical models of MLL-rearranged leukemia

EPZ-5676 is a small molecule inhibitor of the histone methyltransferase DOT1L currently in clinical development and represents a first in class novel therapeutic agent for the treatment of MLL-rearranged (MLL-r) leukemia. In preclinical studies, EPZ-5676 selectively inhibited intracellular histone H3K79 methylation, downstream target gene expression and demonstrated complete tumor regression in a MLL-r leukemia xenograft model. We previously reported synergistic and durable anti-proliferative activity when EPZ-5676 was combined with current AML standard of care drugs, cytarabine and daunorubicin in MLL-r leukemia models MOLM-13 (MLL-AF9) and MV4-11 (MLL-AF4). Combination benefit was also observed when MLL-r cells were treated with cytarabine, prior to co-treatment with EPZ-5676. Additionally, both cytarabine and the DNA methyltransferase inhibitor azacitidine, displayed synergistic anti-leukemic activity in MLL-r rearranged cells in a 7 day co-treatment model (7 days of continuous treatment with EPZ-5676 and second agent; see Klaus et al, JPET, 2014). In this report we discuss results of investigating additional treatment schedules using EPZ-5676 in combination with azacitidine in MLL-r cells. Cells were pretreated with azacitidine at nanomolar concentrations known to reverse promoter DNA-hypermethylation and alter the chromatin state (Tsai et al., Cancer Cell, 2012). We found treating MV4-11 and MOLM-13 cells once daily for three consecutive days followed by sequential treatment with EPZ-5676 elicited a synergistic anti-proliferative effect using the Chou-Talalay method (Chou, Pharmacol Rev., 2006). Results of studies to investigate the mechanism of this synergistic cell killing, including evaluation of differentiation markers and Annexin V staining will be reported. To determine if combinations of EPZ-5676 with cytarabine or azacitidine were tolerable and efficacious in vivo, nude rats implanted subcutaneously with MV4-11 tumors were treated using a range of doses and schedules. Azacitidine and cytarabine were delivered by intraperitoneal injection once daily for 14 days at their respective maximum tolerated doses of 2 and 200 mg/kg. Dosing at the established MTD, these agents inhibited the subcutaneous MV4-11 tumor growth by 50% compared to vehicle controls. Efficacy results from the EPZ-5676 combination studies with cytarabine or azacitidine will be presented. In summary, our results indicate that EPZ-5676 in combination with cytarabine or azacitidine revealed a synergistic effect, regardless of the treatment schedule used in preclinical models of MLL-r leukemia. Tolerable in vivo rat combination doses for EPZ-5676 with both cytarabine and azacitidine have been determined in support of potential future assessment of these combinations in MLL-r leukemia patients. Citation Format: Christine R. Klaus, Scott R. Daigle, Vivek Chopra, Jeffrey A. Keats, Carly T. Campbell, Dorothy Iwanowicz, Edward J. Olhava, Margaret P. Scott, Roy M. Pollock, Robert A. Copeland, Jesse J. Smith, Jorge DiMartino, Stephen J. Blakemore, Alejandra Raimondi. DOT1L inhibitor EPZ-5676 synergizes with cytarabine and azacitidine in preclinical models of MLL-rearranged leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5383. doi:10.1158/1538-7445.AM2015-5383

Abstract B217: A Phase I study of CC-486 (oral azacitidine) to evaluate the pharmacokinetics (PK) and pharmacodynamics (PD) of azacitidine administered alone and in combination with carboplatin or ABI-007 (NAB-paclitaxel) in subjects with solid tumors.

Background: CC-486 is an orally bioavailable formulation of azacitidine that has demonstrated clinical activity in subjects with Myelodysplastic Syndromes (MDS) and Acute Myleoid Leukemia (AML). In these patient populations, CC-486 in extended dosing regimens, 200 mg and 300 mg given daily on a 14 or 21 out of 28-day schedule, induces sustained global genomic hypomethylation. Methods: This Phase I, multicenter, open-label study was conducted in subjects with solid tumors to evaluate: 1) the effect of carboplatin or ABI-007 on the PK of CC-486; 2) the effect of CC-486 on the PK of carboplatin or ABI-007; and 3) the PD (DNA methylation) effects of CC[[Unable to Display Character: ‑]]486 as a single agent and in combination with carboplatin or ABI-007. Safety and efficacy information of this trial are presented in a separate abstract submission. Subjects received CC-486 on Days 1-14 with carboplatin AUC 4 on Day 8 of a 21-day cycle (Arm A), CC-486 on Days 1-14 with ABI-007 100 mg/m2 given weekly starting on Day 8 of a 21-day cycle (Arm B), or CC-486 on Days 1-21 of a 21-day cycle (Arm C). Results: High inter-subject variability was observed in PK concentrations and parameters. In subjects from Arms A and B, following administration of CC-486 alone or in combination with carboplatin or ABI-007, CC-486 was rapidly absorbed and reached Tmax within approximately 1.0 hour post-dose (median); azacitidine PK parameters (AUC, Cmax, half-life elimination, apparent clearance, and volume of distribution) were comparable following administration of CC-486 alone or in combination. Similarly, carboplatin and ABI-007 PK parameters were similar following administration of carboplatin or ABI-007 alone or in combination with CC-486. In subjects from the 3 Arms, global genomic hypomethylation was observed in PBMCs, with maximum effect on Day 15. A PK/PD (AUC/hypomethylation change on Day 15) correlation was noted (r>0.5; p<0.01). Conclusion: Carboplatin and ABI-007 had minimal to no effect on CC-486 PK parameters, and vice-versa. Following administration of CC-486, global genomic hypomethylation was correlated with plasma AUC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B217. Citation Format: Eric Laille*, Aaron N. Nguyen*, Gengxi Chen, Drew Rasco, Patricia LoRusso, Daniel D. Von Hoff, Johanna Bendell, Pamela Munster, William J. Edenfield, Ramesh Ramanathan, Michael B. Gonzalez, Anne Blackwood-Chirchir, Jorge DiMartino. A Phase I study of CC-486 (oral azacitidine) to evaluate the pharmacokinetics (PK) and pharmacodynamics (PD) of azacitidine administered alone and in combination with carboplatin or ABI-007 (NAB-paclitaxel) in subjects with solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B217.

Abstract 4457: The HDAC inhibitor romidepsin prevents the emergence of drug-tolerant cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Drug resistance represents a major obstacle to successful cancer treatment. Various resistance mechanisms have been described (e.g., drug efflux pumps). Recent data points to the pre-existence of a subpopulation of cancer cells termed drug-tolerant persisters (DTPs) that exhibit an epigenetically-mediated tolerance to high concentrations of chemotherapeutic drugs (Sharma, 2010). Although DTPs were largely quiescent, a small fraction resumed growth even in the presence of high drug concentrations, giving rise to drug-tolerant expanded persisters (DTEPs). Levels of histone H3 lysine 4 (H3K4) methylation and H3K14 acetylation were decreased in DTPs and DTEPs, and co-treatment (Q3D) of cancer cells with the anticancer drug in combination with histone deacetylase inhibitors (HDACi) inhibited DTP/DTEP growth, implicating an epigenetic mechanism. Purpose: To assess whether the HDACi romidepsin (ROMI) can prevent the emergence of drug-tolerant cancer cells, to determine the stage at which ROMI acts (killing DTPs or preventing DTEP expansion), and to establish the dose-schedule requirements for the effect. Methods: The M14 melanoma and HCC827 NSCLC cell lines were treated in 24-well plates with AZ628 (B-Raf inhibitor) or Erlotinib (ERL; EGFR inhibitor), respectively, alone or in combination with ROMI, TSA, or azacitidine (AZA). ROMI, TSA, AZ628, and ERL were added every 3 days, while AZA was added daily. Cell viability was monitored periodically over 45 days using CellTiter-Glo. In subsequent experiments, reduced schedules from the “continuous” (Q3D for 45 days) schedule were tested, including Q3D schedules given for 1-8 times. To evaluate minimal daily exposure, cancer cells were exposed to ROMI for only 6 hours. Results: Massive cell death was observed after 9 days of treatment of M14 and HCC827 cells with 100X IC50 concentrations of AZ628 and ERL, respectively. With continued AZ628/ERL treatments, however, the few remaining cells (DTPs) began to proliferate, ultimately yielding a large population of AZ628/ERL-resistant cells (DTEPs). “Continuous” (Q3D for 45 days) co-treatment of cancer cells with AZ628/ERL in combination with sub-lethal concentrations of ROMI and TSA, but not AZA, prevented the emergence of DTEPs. Short (6-hour) exposures to higher ROMI and TSA concentrations on the “continuous” schedule were also sufficient to prevent DTEP growth. Interestingly, “continuous” dosing of HDACi was not required to prevent DTEPs from forming, as reducing the schedule to 2 weeks Q3D had the same effect, suggesting that there is a period of susceptibility to the DTP or early DTEP population. Conclusions: ROMI prevents DTEP emergence. Dosing ROMI for 2 weeks Q3D and 6-hour exposures are sufficient for this effect. These results show that co-treatment with ROMI can prevent drug resistance and provide guidance to the dose and schedule requirements to consider for clinical evaluation in this setting. Citation Format: Manith Norng, Aaron N. Nguyen, Jorge F. DiMartino, Kyle J. MacBeth. The HDAC inhibitor romidepsin prevents the emergence of drug-tolerant cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4457. doi:10.1158/1538-7445.AM2013-4457

Abstract 4656: Development of a robust preclinical model for studying the mechanism of azacitidine priming for platin-induced cytotoxicity.

Background: Chemosensitization by azacitidine (AZA) priming has been demonstrated preclinically and is now supported by clinical data, but the underlying mechanism for the priming effect is not well understood. Previous in vitro studies of priming have focused on individual cell lines and specific epigenetically-silenced genes; however, we show that sensitization is not a universal effect of AZA pretreatment in all cancer cell lines or with all combination agents. Studying the molecular differences between cell lines that are sensitized and those that are not may yield insights into the mechanisms of priming. Purpose: To identify solid cancer cell lines that consistently show AZA sensitization to platin-induced cytotoxicity, for future molecular characterization of the priming mechanism. Methods: Ninety-two solid cancer cell lines, from seven indications, were treated with vehicle or 1μM AZA for 24h before being treated with carboplatin (CARB), cisplatin (CIS), or Abraxane (ABX) for 72h. CellTiter-Glo was used to measure cell viability. The fractional product method and normalized dose-response curves were used to characterize the drug combination interaction. In subsequent experiments, various AZA pretreatment durations (4-72h) were tested. DNA was prepared for DNA methylation (LINE-1) analysis, and cell lysates were harvested for DNMT1 Western blotting. Results: In a subset of cancer cell lines, AZA pretreatment resulted in enhanced sensitivity of cells to CARB and CIS. Seven cell lines across 5 cancer indications consistently showed a sensitization effect with AZA priming, decreasing the CARB IC50 by a mean of 59.6% (range 52.9-68.4%) and CIS IC50 by a mean of 44.6% (range 36.2-54.6%). Priming for CARB was concordant with priming for CIS, but not for ABX, indicating that the AZA pretreatment does not globally increase the susceptibility of cells to cytotoxic agents through non-specific toxic effects; rather, these results suggest that priming by AZA has specific pharmacodynamic effects unique to the combination agent. Follow-on experiments demonstrated that AZA pretreatment of cells for 18-24h was required for priming, whereas the magnitude of priming was not increased with longer (48-72h) AZA pretreatment durations. Effects of AZA on DNMT1 depletion and DNA hypomethylation were detected in all cell lines tested. The magnitude of these proximal PD effects did not correlate with priming, suggesting that differences in sensitization cannot be attributed to differential drug uptake and DNA incorporation. Conclusions: We identified a panel of solid cancer cell lines that consistently demonstrate sensitization to CARB and CIS with AZA priming. By comparing genetic and epigenetic profiles between these lines and a closely matched set of cell lines that does not show priming, we hope to generate clinically testable hypotheses for predictors of sensitization in patients. Citation Format: Aaron N. Nguyen, Manith Norng, Antonio Luna-Moran, Kyle J. MacBeth, Jorge F. DiMartino. Development of a robust preclinical model for studying the mechanism of azacitidine priming for platin-induced cytotoxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4656. doi:10.1158/1538-7445.AM2013-4656