Chiao, Judy H.

Introduction: Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm with limited therapeutic options and dismal outcomes. Truncating mutations in the epigenetic modifier ASXL1 are common and lead to a proliferative disease phenotype, resistance to epigenetic therapies, and higher rates of AML transformation. ASXL1-mutant CMML is characterized by widespread epigenetic remodeling and up-regulation of key leukemogenic driver genes (e.g. HOXA9 and its co-factor MEIS1). There is a genotype-specific activation of enhancers in ASXL1-mutant CMML that is associated with up-regulation of these leukemogenic driver genes. Here, we test the hypothesis that disrupting the oncogenic interactions at these genotype-specific enhancers can be exploited for therapeutic benefit. Methods: We identified activated enhancers specific to ASXL1-mutant CMML as previously described ( Binder et al. Nat Commun 2022;13(1):1434). To disrupt the E2542605 enhancer, we created unique mapped single guide RNAs (confirmed by BLAT). We generated a ribonucleoprotein complex using the Alt-R CRISPR-Cas9 system (IDT Technologies, Coralville IA). We combined the RNP along with Electroporation Enhancer to 3x10^5 cells of the monocytic cell line U937 in 20uL strip-wells provided by the SF Cell Line 4D-Nucleofector X Kit (Lonza, Basel, Switzerland). To deliver the RNP, U937 cells were electroporated via listed cell line specific protocol by the Amaxa 4D-Nucleofector (Lonza). Forty-eight hours post-electroporation, cells were split and sequenced for E2542605 genomic structure disruption. In parallel, RNA was harvested, converted to cDNA and qPCR for HOXA9 and MEIS1 expression was performed using PrimeTime qPCR FAM labeled probe assays (IDT). Gene expression was determined using delta-delta Ct method. For assessment of selective sensitivity of ASXL1 / enhancer interactions, we performed progenitor colony forming assays using primary bone marrow mononuclear cells from CMML patients with or without ASXL1 mutations and healthy normal controls (n=2 in each category), exposed to increasing concentrations of the dual p300/CBP and BET bromodomain inhibitor EP31670. Briefly, we plated cells in methocellulose fortified with recombinant human cytokines supporting hematopoietic growth in duplicate, in a range of EP31670 concentrations. After 10 days, visible hematopoietic colonies were quantified and characterized. IC 50 values were generated using non-linear regression log(EP31670) vs. normalized response. Results: We computationally identified candidate transcriptional regulators enriched at the activated enhancers specific to ASXL1-mutant CMML, including BRD4 (FDR<2.57x10 -370), CBP (FDR=2.56x10 -217), and p300 (FDR=6.36x10 -93). We selected the ASXL1-mutant-specific enhancer E2542605 (predicted to drive HOXA9 expression), given the up-regulation of HOXA9 in ASXL1-mutant CMML (log 2-fold change +2.05, FDR=0.006, n=14). We disrupted E2542605 using two different CRISPR guides, leading to a PCR trace detection of a 14bp deletion and a 26bp indel in the E2542605 enhancer region, respectively. Both enhancer disruptions led to a down-regulation of its putative target gene HOXA9: Mean relative fold-change 0.36 (p=0.001, n=3) and 0.65 (p=0.046, n=3), respectively ( Fig. 1a). We then used a pharmacologic approach (EP31670) to target the candidate transcriptional regulators involved in the oncogenic interaction at these ASXL1-mutant-specific enhancers. We observed selective sensitivity to growth of CMML colonies (particularly ASXL1-mutant colonies) compared to ASXL1-wild type cases and normal bone marrow controls ( Fig. 1b). Conclusions: In CMML patients, truncating ASXL1 mutations are associated with the activation of genotype-specific enhancers and the recruitment of an extended repertoire of transcriptional regulators including BRD4, CBP, and p300. In a model system, the disruption of an enhancer predicted to govern the expression of the leukemogenic driver HOXA9, led to a significant down-regulation of HOXA9 expression. Pharmacologically disrupting the oncogenic interaction at such enhancers with EP31670 in primary patient samples led to significant therapeutic effects at nanomolar drug concentrations in a genotype-specific manner. Oncogenic interactions at activated enhancers are viable therapeutic targets in ASXL1-mutant CMML and warrant further clinical investigation.

BackgroundAcute myeloid leukemia (AML) is fatal in elderly patients who are unfit for standard induction chemotherapy. The objective of this study was to evaluate the survival benefit of administering sapacitabine, an oral nucleoside analogue, in alternating cycles with decitabine, a low‐intensity therapy, to elderly patients with newly diagnosed AML.MethodsThis randomized, open‐label, phase 3 study (SEAMLESS) was conducted at 87 sites in 11 countries. Patients aged ≥70 years who were not candidates for or chose not to receive standard induction chemotherapy were randomized 1:1 to arm A (decitabine in alternating cycles with sapacitabine) received 1‐hour intravenous infusions of decitabine 20 mg/m2 once daily for 5 consecutive days every 8 weeks (first cycle and subsequent odd cycles) and sapacitabine 300 mg twice daily on 3 consecutive days per week for 2 weeks every 8 weeks (second cycle and subsequent even cycles) or to control arm C who received 1‐hour infusions of decitabine 20 mg/m2 once daily for 5 consecutive days every 4 weeks. Prior hypomethylating agent therapy for preexisting myelodysplastic syndromes or myeloproliferative neoplasms was an exclusion criterion. Randomization was stratified by antecedent myelodysplastic syndromes or myeloproliferative neoplasms, white blood cell count (<10 × 109/L and ≥10 × 109/L), and bone marrow blast percentage (≥50% vs <50%). The primary end point was overall survival (OS). Secondary end points were the rates of complete remission (CR), CR with incomplete platelet count recovery, partial remission, hematologic improvement, and stable disease along with the corresponding durations, transfusion requirements, number of hospitalized days, and 1‐year survival. The trial is registered at ClinicalTrials.gov (NCT01303796).ResultsBetween October 2011 and December 2014, 482 patients were enrolled and randomized to receive decitabine administered in alternating cycles with sapacitabine (study arm, n = 241) or decitabine monotherapy (control arm, n = 241). The median OS was 5.9 months on the study arm versus 5.7 months on the control arm (P = .8902). The CR rate was 16.6% on the study arm and 10.8% on the control arm (P = .1468). In patients with white blood cell counts <10 × 109/L (n = 321), the median OS was higher on the study arm versus the control arm (8.0 vs 5.8 months; P = .145), as was the CR rate (21.5% vs 8.6%; P = .0017).ConclusionsThe regimen of decitabine administered in alternating cycles with sapacitabine was active but did not significantly improve OS compared with decitabine monotherapy. Subgroup analyses suggest that patients with baseline white blood cell counts <10 × 109/L might benefit from decitabine alternating with sapacitabine, with an improved CR rate and the convenience of an oral drug. These findings should be prospectively confirmed.

Abstract Background: Sapacitabine is a novel oral nucleoside analogue with a unique ability to induce single-strand DNA breaks after incorporation into DNA, leading to production of double-strand DNA breaks and/or G2 cell cycle arrest. In AML cell lines, the active metabolite of sapacitabine, CNDAC, is synergistic with hypomethylating agents (HMAs) particularly when treated with HMAs first. In a pilot study, there were 6 CRs and 2 PRs in 25 patients treated with sapacitabine in alternating cycles with decitabine. This global randomized phase 3 study evaluated the survival benefit of this treatment strategy vs. decitabine monotherapy. Methods: Decitabine 20 mg/m2 was administered intravenously daily x 5 days of a 4-week cycle (for the control arm and odd cycles of the study arm) alternating with sapacitabine 300 mg p.o. b.i.d. x 3 days/week x 2 weeks of a 4-week cycle (even cycles of the study arm). The safety of these doses was further evaluated in the lead-in phase of the phase 3 study to confirm the findings from the pilot study. Eligible patients were ≥70 years with untreated AML and unsuitable for or unwilling to receive standard induction chemotherapy. Patients who had received HMAs for prior MDS or MPD were excluded. Results: For 482 patients randomized to receive decitabine/sapacitabine (n=241) vs. decitabine only (n=241), randomization was stratified by the presence of antecedent MDS or MPN, peripheral white blood cell count (WBC &lt;10,000 vs. ≥10,000) and bone marrow blast percentage (≥50% vs. &lt; 50%). Median age was 77 years (range 70-90), and 317 patients had de novo AML (66%), 165 secondary AML (34%). WBC was ≥10,000 in 161 patients (33%) and &gt;40,000 in 59 patients (12%); 194 patients (40%) had unfavorable cytogenetic risk by SWOG criteria. Disease characteristics were well balanced in both arms. In total, 13.7% of patients achieved CR, more on the study arm vs. control (16.6% vs. 10.8%). A total of 37.3% treated patients received ≥5 cycles of treatment, similar on both arms, as were 30- and 60-day death rates. Median overall survival was 5.9 months on the study arm vs. 5.7 months on control arm, which did not reach a statistically significant difference. In the subgroup of patients with &lt;10,000 WBC (n=321), median overall survival was higher on the study arm vs. control arm (8.0 months vs. 5.8 months), as was CR rate (21.5% vs. 8.6%). Grade 3 or higher adverse events (regardless of causality) that occurred in &gt;10% patients were thrombocytopenia, anemia, neutropenia, pneumonia, febrile neutropenia, sepsis, and disease progression. Conclusion: The regimen of sapacitabine administered in alternating cycles with decitabine was active and well tolerated but it did not significantly improve overall survival as compared to decitabine monotherapy. Further analyses are being conducted to characterize the subgroups of patients who appeared to have benefited from this treatment regimen and the potential cost savings associated with the use of an oral drug. Disclosures Kantarjian: Amgen: Research Funding; Pfizer: Research Funding; Delta-Fly Pharma: Research Funding; ARIAD: Research Funding; Bristol-Meyers Squibb: Research Funding; Novartis: Research Funding. Goldberg: Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; Celgene: Speakers Bureau; COTA: Employment, Equity Ownership; Pfizer: Honoraria; Ariad: Speakers Bureau; Jazz: Speakers Bureau. Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees. Strickland: Boehringer Ingelheim: Consultancy; Daiichi Sankyo: Consultancy; CTI BioPharma: Consultancy; Tolero Pharmaceuticals: Consultancy; Sunesis Pharamaceuticals: Consultancy, Research Funding; Baxalta: Consultancy; Alexion Pharmaceuticals: Consultancy; Astellas Pharma: Honoraria. Rubenstein: Alexion Pharmaceuticals: Speakers Bureau. Arellano: Cephalon Oncology: Research Funding. Schiller: bluebird bio: Research Funding; mateon therapeutics: Research Funding. Gaidano: Roche: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Montesinos: Celgene Corporation: Honoraria, Research Funding. Rizzieri: Shire: Research Funding; Erytech: Research Funding. Subramanian: Boehringer-Ingelheim, Lilly, BMS, Astra-Zeneca, Pfizer, Biocept: Consultancy, Speakers Bureau. Buyse: IDDI: Employment, Equity Ownership. Chiao: Cyclacel LTD: Employment, Equity Ownership.