Huw, Ling‐Yuh

Abstract Idasanutlin, an MDM2 antagonist, showed clinical activity and a rapid reduction in JAK2 V617F allele burden in patients with polycythemia vera (PV) in a phase 1 study. This open-label phase 2 study evaluated idasanutlin in patients with hydroxyurea (HU)-resistant/-intolerant PV, per the European LeukemiaNet criteria, and phlebotomy dependence; prior ruxolitinib exposure was permitted. Idasanutlin was administered once daily on days 1 through 5 of each 28-day cycle. The primary end point was composite response (hematocrit control and spleen volume reduction > 35%) in patients with splenomegaly and hematocrit control in patients without splenomegaly at week 32. Key secondary end points included safety, complete hematologic response (CHR), patient-reported outcomes, and molecular responses. All patients (n = 27) received idasanutlin; 16 had response assessment (week 32). Among responders with baseline splenomegaly (n = 13), 9 (69%) attained any spleen volume reduction, and 1 achieved composite response. Nine patients (56%) achieved hematocrit control, and 8 patients (50%) achieved CHR. Overall, 43% of evaluable patients (6/14) showed a ≥50% reduction in the Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score (week 32). Nausea (93%), diarrhea (78%), and vomiting (41%) were the most common adverse events, with grade ≥ 3 nausea or vomiting experienced by 3 patients (11%) and 1 patient (4%), respectively. Reduced JAK2 V617F allele burden occurred early (after 3 cycles), with a median reduction of 76%, and was associated with achieving CHR and hematocrit control. Overall, the idasanutlin dosing regimen showed clinical activity and rapidly reduced JAK2 allele burden in patients with HU-resistant/- intolerant PV but was associated with low-grade gastrointestinal toxicity, leading to poor long-term tolerability. This trial was registered at www.clinincaltrials.gov as #NCT03287245.

Background Activating mutations in JAK2 are the molecular hallmark of PV. However, achieving molecular remission is not a validated endpoint in PV, as its correlation with patient-relevant endpoints, such as thrombotic events or disease progression, is not well established. The expression of MDM2, a negative regulator of wild-type p53, is significantly higher in CD34+ cells from patients with PV vs those from healthy controls. Therefore, MDM2 antagonists such as idasanutlin (idasa) may have selective and potentially disease-modifying effects in PV. This exploratory analysis reports baseline mutational profiling and serial measurements of JAK2 V617F variant allele frequency (VAF) from patients treated with idasa in the Phase II NP39761 study (NCT03287245). Methods Patients with PV, as defined by the 2016 World Health Organization criteria, were eligible for inclusion if their disease was HU resistant/intolerant according to European LeukemiaNet criteria and they were phlebotomy dependent (≥ 1 phlebotomy in the 16-wk period prior to screening). Prior PV therapy with ruxolitinib and/or interferon was allowed. Idasa was given orally once daily on days 1-5 of 28-day cycles for up to 24 mo. The starting dose was 150 mg, but dose increase (to 200 mg) or decrease (to 100 mg) was allowed per protocol-defined criteria. The primary efficacy endpoints were hematocrit control (HTC), defined as maintaining acceptable hematocrit levels with ≤ 1 phlebotomy between cycle 1, day 1 (C1D1) and wk 8 and no phlebotomy after wk 8, and complete hematologic remission (CHR), defined as HTC together with platelets < 400 × 109/L and white blood cell count < 109/L on the day of response assessment (or most recent test). JAK2 V617F VAF was analyzed at screening and at C3D28, C5D28 and wk 32. The JAK2 V617F VAF was determined using quantitative polymerase chain reaction. Baseline blood samples were sequenced using the Foundation Medicine FoundationOne Heme next-generation sequencing panel, which interrogates > 400 DNA-sequenced genes, to explore the genetic landscape of PV beyond JAK2 V617F. Results All 27 patients enrolled in the study harbored JAK2 V617F. Baseline and ≥ 1 later JAK2 VAF measurements were available in 23 patients, allowing for analysis. The median age was 56 y (range, 34-74), and 7 patients (26%) had been previously treated with ruxolitinib. Median treatment duration was 257 days (range, 5-677), and the median number of treatment cycles was 8 (range, 1-22). JAK2 V617F VAFs at baseline were highly variable, ranging from 7% to 96% (median, 66%). A reduction in JAK2 V617F VAF was observed as early as after 3 cycles (median reduction, 39%; n = 19) and was sustained in patients receiving treatment, with a median reduction of 58% at C5D28 (n = 17) and of 76% at wk 32 (n = 13). The median reductions in JAK2 V617F VAF were significantly higher for patients with CHR and HTC than in nonresponders on C3D28 (CHR, P < 0.01; HTC, P = 0.04) and C5D28 (CHR, P < 0.01; HTC, P = 0.03), with a similar trend also at wk 32 (CHR, P = 0.06; HTC, P = 0.19) (Figures). Baseline targeted sequencing (n = 16) identified several additional subclonal mutations. However, no TP53 mutations were detected. Variants in TET2 and ASXL1 were detected in 37% (6 of 16) and 20% (3 of 16) of patients, with VAFs of 2%-51% and 38%-50%, respectively. Notably, 68% of the patients had variants detected in 1 to 3 DNA repair genes, including ATM (VAF, 47%-52%), ATR (VAF, 49%), BRCA2 (VAF, 47%-51%), PARP1 (VAF, 52%), PARP3 (VAF, 48%), FANCM (VAF, 47%-50%), BLM (VAF, 48%) and MSH3 (VAF, 49%). In the absence of sequencing of patient-matched non-hematologic tissue and the VAFs being ≈50%, it is possible some of these variants are germline. Whether such variants predispose to PV is unknown. Conclusions Idasa has relevant clinical activity in patients with PV who are resistant to or intolerant of HU and showed rapid JAK2 V617F VAF reduction in a subset of patients as early as after 3 treatment cycles. The median JAK2 VAF reduction was significantly greater in patients who achieved CHR and HTC after 3 and 5 cycles of idasa treatment, suggesting that early molecular response was associated with higher likelihood of clinical response. Baseline mutations were seen in several genes commonly mutated in PV. A high frequency of variants in DNA repair genes has not previously been reported in PV and may contribute to genomic instability in this disease. Figure Disclosures Passamonti: Roche: Other: Support of parent study and funding of editorial support; BMS: Speakers Bureau; Novartis: Speakers Bureau. El-Galaly:F. Hoffmann-La Roche: Current Employment, Other: Support of parent study and funding of editorial support. Gerds:Celgene: Consultancy, Research Funding; Gilead Sciences: Research Funding; CTI Biopharma: Consultancy, Research Funding; Imago Biosciences: Research Funding; AstraZeneca/MedImmune: Consultancy; Roche/Genentech: Research Funding; Incyte Corporation: Consultancy, Research Funding; Pfizer: Research Funding; Apexx Oncology: Consultancy; Sierra Oncology: Research Funding. Gupta:Incyte: Honoraria, Research Funding; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy. Higgins:Roche: Current Employment, Current equity holder in publicly-traded company, Other: Support of parent study and funding of editorial support. Huw:Roche: Current Employment, Other: Support of parent study and funding of editorial support. Maffioli:Roche: Other: Support of parent study and funding of editorial support. Mesa:Sierra Oncology: Consultancy; Incyte: Research Funding; Samus Therapeutics: Research Funding; Promedior: Research Funding; Genentech: Research Funding; Novartis: Consultancy; CTI BioPharma: Research Funding; LaJolla Pharmaceutical Company: Consultancy; AbbVie: Research Funding; Bristol Myers Squibb: Research Funding. Rambaldi:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support of parent study and funding of editorial support. Received travel support., Research Funding; Sanofi: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Astellas: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support from Gilead.; BMS/Celgene: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company). Advisory board and speaker fees from Pfizer.; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company). Research grant from Amgen Inc.; Omeros: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); University of Milan: Current Employment. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint: Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Yacoub:Incyte: Speakers Bureau; Hylapharm: Current equity holder in private company; Cara Therapeutics: Current equity holder in publicly-traded company; Dynavax: Current equity holder in publicly-traded company; Agios: Honoraria, Speakers Bureau; Novartis: Speakers Bureau; Roche: Other: Support of parent study and funding of editorial support; Ardelyx: Current equity holder in publicly-traded company. Mascarenhas:Celgene, Prelude, Galecto, Promedior, Geron, Constellation, and Incyte: Consultancy; Incyte, Kartos, Roche, Promedior, Merck, Merus, Arog, CTI Biopharma, Janssen, and PharmaEssentia: Other: Research funding (institution).

Abstract Extensive biological and clinical evidence supports the notion many CRC tumors are addicted to EGFR/KRAS/MAPK signaling. For example, mutations in KRAS, BRAF and other MAPK pathway components confer intrinsic or acquired resistance to anti-EGFR therapy. However, a significant proportion of acquired resistance cases have remained unexplained. ARID1A mutations are found in approximately 11% of CRC tumors, but a connection between ARID1A mutations and EGFR/MAPK signaling activation in CRC has not been previously suspected. By leveraging the genomic biomarker data collected from 333 patients at baseline and at progression who participated in the CALGB/SWOG 80405 trial, we found that mutations in ARID1A, a key component of SWI/SNF complex, were enriched in the cell free DNA of 6/16 of the patients whose tumors had become resistant to anti-EGFR therapy with ARID1A mutations detected at either timepoint (adj. p = 0.03, OR = 0.09, two-tailed fisher's exact test). In contrast, there was no evidence of enriched ARID1A mutations in patients treated with bevacizumab. To investigate the potential role of a broader ARID1A deficiency phenotype in resistance to cetuximab therapy, we developed an ARID1A mutant-like signature from TCGA that captures the transcriptional profile characteristic of ARID1A mutants. Using this signature to stratify CALGB patients based on gene expression data collected on tissue at diagnosis, we also found patients with an ARID1A mutant-like gene expression profile had worst outcome in patients treated with cetuximab than bevacizumab with adjustment for established clinical variables for both OS [p = 0.0002, hazard ratio (HR) 6.2, 95% confidence interval (CI) 2.4-16], and PFS [p = 0.0008, HR 4.7 (1.9 -12)], suggesting that ARID1A mutations may be also a mechanism of intrinsic resistance to anti-EGFR therapies. Gene expression analysis demonstrated that ARID1A deficiency leads to re-activation of an EGFR-like signature, suggesting reactivation of this pathway as a possible mechanism of resistance. Furthermore, CRISPR knockout of ARID1A in the cetuximab-sensitive CRC cell line NCI-H508 conferred elevated MAPK signaling relative to parental line and resistance to cetuximab in culture. Consistent with these findings, we also observed strong mutual exclusivity between ARID1A mutations and mutations in the EGFR/MAPK signaling pathway in more than 40,000 lung and colorectal cancer patients profiled in the FoundationCore® database. Strikingly, in lung cancers where EGFR mutations structural variants (SV) are more prevalent, EGFR SV was the top mutually exclusive alteration with ARID1A mutations in lung cancers. Of 5980 lung cancer patients with ARID1A and/or EGFR SVs, only 100 patients (1.7%) had both SVs. Taken together, our data suggest that ARID1A loss-of-function mutations may promote resistance to cetuximab by driving an EGFR-like transcription program in the absence of ligand-dependent activation of EGFR. Our results suggest that ARID1A defects could be potentially used as an exclusion biomarker for cetuximab treatment decisions, and they provide a rationale for exploring therapeutic MAPK inhibition in ARID1A mutant CRC patients. Citation Format: Radia Marie Johnson, Xueping Qu, Joshua Thomas, Yvonne Kschonsak, Ling-Yuh Huw, Fang-Shu Ou, Ethan Sokol, Nnamdi Ihuegbu, Oliver Zill, Omar Kabbarah, Anneleen Daemen, Richard Bourgon, Alan Venook, Federico Innocenti, Heinz-Josef Lenz, Felipe de Sousa e Melo, Carlos Bais. ARID1A mutations induce an EGFR-like gene expression signature and confer intrinsic and acquired resistance to cetuximab treatment in first line metastatic CRC [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 LB-063.