Micol, Jean‐Baptiste

Background: Heterozygous somatic mutations in the genes encoding RNA splicing factors SF3B1, U2AF1, SRSF2 or ZRSR2 induce aberrant splicing in cancer cells and are among the most common mutations in patients with MDS, AML or CMML. H3B-8800 is an orally available small molecule that binds to the SF3b complex and induces alternative splicing changes in cells. Because splicing factor mutant cells depend on residual wild-type function of splicing factors for survival, we hypothesized that H3B-8800 would induce preferential cell killing of mutant cells by further perturbing splicing to synthetic lethality. In pre-clinical models, H3B-8800 preferentially kills spliceosome mutant cells and induces antitumor activity in xenograft leukemia models with core spliceosome mutations (Seiler M et al Nature Medicine 2018). Therefore, we conducted a phase I clinical trial (NCT02841540) of H3B-8800 in patients with MDS, AML or CMML. Here we describe the safety profile and clinical outcomes of the dose escalation cohorts in this phase I trial. Methods: This phase I trial explored the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of H3B-8800 in patients with myeloid cancers. Dose escalation cohorts, employing a standard 3+3 design, examined 2 different once daily dosing regimens (schedule I: 5 days on/9 days off; schedule II: 21 days on/7 days off) in a 28-day cycle, with stratification based on lower-risk (LR) versus higher-risk (HR) myeloid neoplasms. Results: As of June 16th, 2019, 84 patients were enrolled at 24 centers in the US and Europe. Dose ranged from 1-40 mg among 65 patients on schedule I, while 19 patients were enrolled with dose ranging from 7-20 mg on schedule II. The patient population included AML (n=38), CMML (n=4), HR-MDS (n=20), LR-MDS (n=21) and 1 MDS with unknown risk level. Most patients (88%) had spliceosome mutations of interest. The most common mutations were in SRSF2 (p.P95H in 17, p.P95L in 9, p.P95_R102Del in 4), SF3B1 (p.K700E in 11, p.R625C in 4), and U2AF1 (p.Q157P in 6, p.S34F in 4). Patients remained on treatment from 7 to 819 days; 25 patients (30%) had time on treatment greater than 180 days, 20% more than 1 year and 2% over 2 years. The median therapy duration for LR-CMML/MDS, HR-CMML/MDS, and AML patients were 216, 62, and 47 days respectively. Most observed treatment related treatment-emergent adverse events (TEAEs) were Grade 1 or 2. The most common treatment-related (as judged by the investigator, >10% frequency) TEAEs in the patients treated on schedule I were diarrhea (75%), nausea (37%), fatigue (28%) and vomiting (27%). The most common treatment-related TEAEs in the patients treated on schedule II were diarrhea (68%), vomiting (42%), QTc prolongation (21%), nausea (16%), and fatigue (16%). The most common dose limiting toxicity was prolongation of the QTcF interval >500 msec (n=2, 40 mg on schedule I and n=1, 20 mg on schedule II, all ≥Grade 3) and bradycardia without other arrhythmias (n=1, 14 mg on schedule II, ≥Grade 3). No ophthalmic AEs were observed; 1 patient (LR-MDS) experienced durable marrow aplasia. The maximum tolerated dose (MTD) has not been confirmed for either Schedule I or Schedule II. PK analysis indicates that H3B-8800 is rapidly absorbed and exhibits dose-proportional increase in plasma exposure. Consistent dose-dependent target engagement (i.e., alteration in mature mRNA transcripts) was observed in blood mononuclear cells from patients enrolled in the 2 mg up to 40 mg dose cohorts on both schedules. Despite this splicing modulation, no objective complete responses (CR) or partial responses (PR) meeting International Working Group criteria were observed. One patient with CMML had a durable platelet response that began in Cycle 1 and persisted through Cycle 13. Nine red blood cell (RBC) transfusion-dependent patients with MDS or CMML and 2 patients with AML did not require RBC transfusions for ≥8 weeks (up to 28 weeks) while on study. One platelet transfusion-dependent patient with LR-MDS did not require platelet transfusions for ≥8 weeks. Conclusion: Results from this first-in-human multicenter prospective clinical trial of a splicing modulator in myeloid neoplasms demonstrate dose-dependent target engagement and predictable PK profile of H3B-8800, and safety even with prolonged dosing. Although no objective CR or PR were achieved, decreased RBC or platelet transfusion requirements were observed in 12 (14%) of enrolled patients. Disclosures Steensma: H3 Biosciences: Other: Research funding to institution, not investigator.; Pfizer: Consultancy; Aprea: Research Funding; Stemline: Consultancy; Arrowhead: Equity Ownership; Summer Road: Consultancy; Astex: Consultancy; Onconova: Consultancy. Wermke:Novartis: Honoraria, Research Funding. Greenberg:Notable Labs: Research Funding; Celgene: Research Funding; Genentech: Research Funding; H3 Biotech: Research Funding; Aprea: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees. Font:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Komrokji:Agios: Consultancy; JAZZ: Speakers Bureau; DSI: Consultancy; JAZZ: Consultancy; Incyte: Consultancy; Novartis: Speakers Bureau; celgene: Consultancy; pfizer: Consultancy. Yang:Agios: Consultancy; AstraZeneca: Research Funding. Brunner:Astra Zeneca: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Forty Seven Inc: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding. Ades:Agios: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Helsinn Healthcare: Membership on an entity's Board of Directors or advisory committees. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding. Coombs:H3 Biomedicine: Research Funding. Foran:Agios: Honoraria, Research Funding. Garcia-Manero:Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Amphivena: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Micol:AbbVie: Consultancy; Jazz Pharmaceuticals: Consultancy. Perez De Oteyza:Celgene: Speakers Bureau. Wang:Abbvie: Other: Advisory role; Kite: Other: Advisory role; Jazz: Other: Advisory role; Astellas: Other: Advisory role, Speakers Bureau; celyad: Other: Advisory role; Pfizer: Other: Advisory role, Speakers Bureau; Stemline: Other: Advisory role, Speakers Bureau; Daiichi: Other: Advisory role; Amgen: Other: Advisory role; Agios: Other: Advisory role. Watts:Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Buonamici:H3 Biomedicine: Employment. Kim:H3 Biomedicine: Employment. Gourineni:H3 Biomedicine: Employment. Marino:H3 Biomedicine: Employment. Rioux:H3 Biomedicine: Employment. Schindler:H3 Biomedicine: Employment. Smith:H3 Biomedicine: Employment. Yao:H3 Biomedicine: Employment. Yuan:Eisai: Employment. Yu:H3 Biomedicine: Employment. Platzbecker:Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. OffLabel Disclosure: H3B-8800 (experimental, unapproved)

Abstract Histiocytic neoplasms are clonal, hematopoietic disorders characterized by an accumulation of abnormal monocyte-derived dendritic cells or macrophages in Langerhans Cell (LCH) and non-Langerhans (non-LCH) histiocytoses, respectively. The discovery of the BRAF V600E mutation in ~50% of patients with LCH and the non-LCH Erdheim-Chester Disease (ECD) provided the first molecular target in these patients and novel insights into the pathogenesis of these disorders. However, recurrent mutations in the majority of the ~50% of BRAF V600E-wild type patients with non-LCH are unknown. Moreover, recurrent mutations outside of the MAP kinase pathway are undefined throughout histiocytic neoplasms. To address these issues, we performed whole exome sequencing (WES) of frozen biopsies from 24 patients with LCH (n=10) or ECD (n=14) paired with peripheral blood mononuclear cells. 13/24 patients also underwent RNA sequencing (RNA-seq). All mutations in activating kinases were validated by droplet-digital PCR, while targeted-capture next-generation sequencing validated all others. Both adult (n=18; n=2 with LCH) and pediatric cases (n=9; n=8 with LCH) were included. Using combined WES/RNA-seq, activating kinase alterations were identified in 100% of patients. In LCH, 60% and 40% had BRAF V600E and MAP2K1 mutations, respectively. In non-LCH 51%, 14%, 14%, and 7% were BRAFV600E, ARAF, MAP2K1, and NRAS mutant (Fig1A). Overall, a mean of 7 non-synonymous mutations per adult patient was identified (range 1-22) compared with 5 mutations per pediatric patient (range 4-9; p =ns). Mutations affecting diverse cellular processes were found to co-exist with kinase mutations including mutations in epigenetic modifiers and the p38/MAPK pathway. In addition to kinase point mutations, RNA-seq identified recurrent, in-frame kinase fusions-a first for these disorders. All identified fusions were validated using FISH and RT-PCR. This includes novel fusions in BRAF (RNF11-BRAF and CLIP2-BRAF), as well as therapeutically important fusions in ALK (2 separate KIF5B-ALK fusions) and NTRK1 (LMNA-NTRK1;Fig1B). Expression of each fusion in Ba/F3 cells conferred cytokine-independent growth. Importantly, the BRAF fusions were found to be sensitive to MEK inhibition but resistant to vemurafenib while the ALK fusions conferred sensitivity to the ALK inhibitors crizotinib or alectinib. We next interrogated a validation cohort of 37 BRAF V600E-wild type, non-LCH, formalin-fixed, paraffin-embedded tissue samples using targeted mutational profiling for MAP2K1, ARAF, NRAS, KRAS, and PIK3CA. This revealed activating mutations in MAP2K1 (32%; n=12), NRAS (16%; n=6), KRAS (11%; n=4), PIK3CA (8%; n=3), and ARAF (3%; n=1). Three of the investigated non-LCH patients with refractory disease and progressive organ dysfunction were treated with targeted therapies based on the discovery of novel kinase alterations described above. Treatment of 2 refractory MAP2K1- mutant, non-LCH patients with MEK inhibitors (trametinib or cobimetinib) resulted in dramatic clinical improvement (Fig1C). Both patients have been maintained on MEK inhibitor single-agent therapy with a sustained clinical response for >100 days. Further evidence of effective targeted inhibition was found in a refractory ECD patient carrying an ARAF S214A mutation. This patient failed to respond to 3 lines of prior therapies and suffered near blindness due to disease infiltration in the retina and optic nerves. Given a recent report of complete response to sorafenib in a lung cancer patient with an ARAF S214C mutation, we initiated sorafenib. Within 12 weeks, there was improvement in the patientÕs eyesight and decreased infiltrative disease, coinciding with >50% decrease in mutant ARAF DNA in plasma cell-free DNA. Whole exome and transcriptome sequencing identified activating kinase mutations or translocations in all patients with the common downstream effect of activating the MAPK pathway. The preliminary, dramatic, clinical efficacy observed with use of MEK and RAF inhibitors in MAP2K1 - and ARAF-mutated, non-LCH patients further supports the central role of targeting the MAPK pathway in these tumors. The discovery of the discussed mutations and fusions in diverse kinases provides critical new insights into the genetic events central to a spectrum of adult and pediatric histiocytic neoplasms. Figure 1. Figure 1. Disclosures Off Label Use: This abstract describes use of MEK inhibitors (both tremetinib and cobimetinib) as well as sorafenib for MEK1 and ARAF mutant histiocytosis. . Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc.: Employment, Equity Ownership. Ross:Foundation Medicine Inc.: Employment. Ali:Foundation Medicine Inc.: Employment. Hyman:Chugai Pharma: Consultancy; Biotherapeutics: Consultancy; Atara: Consultancy, Honoraria.

Abstract Histiocytic neoplasms are clonal, hematopoietic disorders characterized by an accumulation of abnormal, monocyte-derived dendritic cells or macrophages in Langerhans cell histiocytosis (LCH) and non-Langerhans cell histiocytosis (non-LCH), respectively. The discovery of BRAFV600E mutations in approximately 50% of these patients provided the first molecular therapeutic target in histiocytosis. However, recurrent driving mutations in the majority of patients with BRAFV600E–wild-type non-LCH are unknown, and recurrent cooperating mutations in non-MAP kinase pathways are undefined for the histiocytic neoplasms. Through combined whole-exome and transcriptome sequencing, we identified recurrent kinase fusions involving BRAF, ALK, and NTRK1, as well as recurrent, activating MAP2K1 and ARAF mutations in patients with BRAFV600E–wild-type non-LCH. In addition to MAP kinase pathway lesions, recurrently altered genes involving diverse cellular pathways were identified. Treatment of patients with MAP2K1- and ARAF-mutated non-LCH using MEK and RAF inhibitors, respectively, resulted in clinical efficacy, demonstrating the importance of detecting and targeting diverse kinase alterations in these disorders. Significance: We provide the first description of kinase fusions in systemic histiocytic neoplasms and activating ARAF and MAP2K1 mutations in non-Langerhans histiocytic neoplasms. Refractory patients with MAP2K1- and ARAF-mutant histiocytoses had clinical responses to MEK inhibition and sorafenib, respectively, highlighting the importance of comprehensive genomic analysis of these disorders. Cancer Discov; 6(2); 154–65. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 109