No abstract

Background: CD123 is a subunit of the interleukin 3 (IL3) receptor and is expressed on the surface of blasts in most cases of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). CD123 expression is often higher on leukemia cells than normal progenitors and may be enriched in residual cells surviving chemotherapy. Uniformly high CD123 is characteristic of blastic plasmacytoid dendritic cell neoplasm (BPDCN), an uncommon leukemia related to AML. Tagraxofusp (TAG, SL-401) is a recombinant protein drug consisting of IL3 fused to a truncated diphtheria toxin payload that targets CD123. Single agent TAG is approved for treatment of BPDCN. In preclinical models and in patients who received TAG, we previously found that TAG resistance in AML and BPDCN was mediated by DNA methylation and downregulation of diphthamide genes (e.g. DPH1) and subsequent resistance to diphtheria toxin (Togami, JCI 2019). TAG resistance was reversible by the hypomethylating agent azacitidine (AZA), and TAG plus AZA was more effective than either alone in xenograft models. Therefore, we performed a phase 1b trial combining TAG with AZA or with AZA and venetoclax (VEN) in patients with AML, MDS, or BPDCN. Methods: Detection of CD123 on blasts by flow or IHC was required. Eligibility included albumin ≥3.2 g/dL, normal cardiac ejection fraction, and hospitalization in cycle 1 to mitigate risks of capillary leak syndrome (CLS). The study followed a 3+3 dose escalation, plus expansion cohorts, of 28-day cycles of TAG with fixed doses of AZA or AZA-VEN. First, we tested 5 schedules of the doublet TAG (5 or 7 μg/kg/d1-5, or 7, 9, or 12 μg/kg/d1-3) with standard doses of AZA (75 mg/m2 d1-7) in newly diagnosed AML (1L), relapsed/refractory AML (R/R), or MDS with ≥10% blasts. Next, we tested 3 doses of the triplet TAG (7, 9, or 12 μg/kg/d4-6) with AZA (75 mg/m2 d1-7) and VEN (400 mg d1-21, ramp up d1-3 in cycle 1) in 1L AML, R/R AML, or R/R BPDCN. Results: 34 patients have been enrolled and 33 are evaluable (one withdrew prior to therapy). The age range is 21-86 (median 67). Median follow up is 8.4 months [95% CI, 7.1-19.1]. 18 received TAG-AZA (5 1L AML, 9 R/R AML, 4 MDS); 15 received TAG-AZA-VEN (9 1L AML, 3 R/R AML, 3 R/R BPDCN). An MTD was not reached, and the recommended phase 2 dose of TAG was determined to be 12 μg/kg/d x3d in each combination. Adverse events (AEs) were as expected for TAG or AZA+/-VEN. Grade 3+ AEs in >10% included neutropenia (30%), thrombocytopenia (27%), anemia (18%), febrile neutropenia (18%), ALT increase (12%), and bilirubin increase (12%). Eleven of 33 (33%) experienced CLS: 8 (24%) were grade 2, 2 grade 3 (6%), and 1 grade 4 (3%). One treatment-related death occurred in a 79 year-old with AML receiving TAG-AZA-VEN who experienced tumor lysis syndrome followed by CLS and multiorgan system failure during cycle 1. Eight of 9 patients (89%) with previously untreated AML (ages 59-81, median 74; 7 of 9 ELN adverse risk) who received TAG-AZA-VEN achieved best response of complete response (CR, n=5) or complete response with incomplete count recovery (CRi, n=3), at TAG doses of 7 (n=2), 9 (n=1), or 12 μg/kg/d x3d (n=6). The 8 with CR/CRi included two AMLs with TP53 mutation and adverse karyotype, and three with secondary AML, two which had concomitant mature pDC expansion, a.k.a. "pDC-AML" (Xiao, Blood 2021). At the time of data cut off, 4 of 8 have gone to allogeneic stem cell transplantation (alloSCT) and 3 remain on therapy. Three patients with relapsed/refractory BPDCN (all had prior single agent TAG) received TAG-AZA-VEN and 2 of 3 responded (CRc=1, CRi=1), who both went to alloSCT. Four patients with previously untreated MDS and ≥10% blasts received TAG-AZA. 3 of 4 responded (all 3 with TP53 mutation) with CR (n=2) and marrow CR (n=1), and two went to alloSCT. Among 14 patients with 1L (5) or R/R (9) AML on TAG-AZA, and 3 with R/R AML on TAG-AZA-VEN, one with 1L AML (17p- and complex karyotype) achieved a CRi on TAG-AZA. Responses in all groups included patients with high and low CD123 by central flow. Conclusions: Combining TAG with AZA or AZA-VEN is feasible, with expected TAG or AZA+/-VEN toxicities. CLS is an important consideration for TAG, requiring monitoring and early implementation of supportive care. The activity of TAG-AZA-VEN in previously untreated AML and R/R BPDCN, and of TAG-AZA in MDS are encouraging, including in high-risk AML/MDS subtypes such as TP53 mutated. These data support continued development of TAG combinations in CD123+ malignancies. Figure 1 Figure 1. Disclosures Lane: Qiagen: Consultancy, Honoraria; N-of-One: Consultancy, Honoraria; Stemline Therapeutics: Research Funding; AbbVie: Research Funding. Stein: Amgen: Consultancy, Speakers Bureau; Celgene: Speakers Bureau; Stemline: Speakers Bureau. Garcia: Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; AstraZeneca: Research Funding; Genentech: Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Prelude: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Stone: AbbVie: Consultancy; Syros: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; Agios: Consultancy, Research Funding; Celgene: Consultancy; Astellas: Membership on an entity's Board of Directors or advisory committees; BerGen Bio: Membership on an entity's Board of Directors or advisory committees; Boston Pharmaceuticals: Consultancy; Bristol Myers Squibb: Consultancy; Foghorn Therapeutics: Consultancy; Elevate Bio: Membership on an entity's Board of Directors or advisory committees; Onconova: Consultancy; Syndax: Membership on an entity's Board of Directors or advisory committees; Syntrix/ACI: Membership on an entity's Board of Directors or advisory committees; Arog: Consultancy, Research Funding; Gemoab: Membership on an entity's Board of Directors or advisory committees; Actinium: Membership on an entity's Board of Directors or advisory committees; Innate: Consultancy; GlaxoSmithKline: Consultancy; Janssen: Consultancy; Jazz: Consultancy; Novartis: Consultancy, Research Funding; Aprea: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Macrogenics: Consultancy. Winer: Novartis: Consultancy; Takeda: Consultancy; Abbvie: Consultancy. Mughal: Oxford University Press, Informa: Other: financial benefit and/or patents ; Stemline: Current Employment, Current holder of stock options in a privately-held company. Brooks: Stemline Therapeutics: Current Employment. Konopleva: KisoJi: Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Rafael Pharmaceuticals: Other: grant support, Research Funding; Sanofi: Other: grant support, Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Ascentage: Other: grant support, Research Funding; Stemline Therapeutics: Research Funding; Agios: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Ablynx: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; Cellectis: Other: grant support; Calithera: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Forty Seven: Other: grant support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding. Pemmaraju: DAVA Oncology: Consultancy; Springer Science + Business Media: Other; Samus: Other, Research Funding; MustangBio: Consultancy, Other; Incyte: Consultancy; Clearview Healthcare Partners: Consultancy; LFB Biotechnologies: Consultancy; Stemline Therapeutics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Abbvie Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Blueprint Medicines: Consultancy; Roche Diagnostics: Consultancy; Novartis Pharmaceuticals: Consultancy, Other: Research Support, Research Funding; Dan's House of Hope: Membership on an entity's Board of Directors or advisory committees; Plexxicon: Other, Research Funding; Celgene Corporation: Consultancy; ASCO Leukemia Advisory Panel: Membership on an entity's Board of Directors or advisory committees; HemOnc Times/Oncology Times: Membership on an entity's Board of Directors or advisory committees; Affymetrix: Consultancy, Research Funding; ASH Communications Committee: Membership on an entity's Board of Directors or advisory committees; Protagonist Therapeutics, Inc.: Consultancy; Sager Strong Foundation: Other; Daiichi Sankyo, Inc.: Other, Research Funding; Cellectis S.A. ADR: Other, Research Funding; Aptitude Health: Consultancy; CareDx, Inc.: Consultancy; Bristol-Myers Squibb Co.: Consultancy; ImmunoGen, Inc: Consultancy; Pacylex Pharmaceuticals: Consultancy.

Fanconi anemia (FA) is a rare disease characterized by congenital defects, bone marrow failure, and atypically early-onset cancers. The FA proteins function cooperatively to repair DNA interstrand crosslinks. A major step in the activation of the pathway is the monoubiquitination of the FANCD2 and FANCI proteins, and their recruitment to chromatin-associated nuclear foci. The regulation and function of FANCD2 and FANCI, however, is poorly understood. In addition, how chromatin state impacts pathway activation is also unknown. In this study, we have examined the influence of chromatin state on the activation of the FA pathway. We describe potent activation of FANCD2 and FANCI monoubiquitination and nuclear foci formation following treatment of cells with the histone methyltransferase inhibitor BRD4770. BRD4770-induced activation of the pathway does not occur via the direct induction of DNA damage or via the inhibition of the G9a histone methyltransferase, a mechanism previously proposed for this molecule. Instead, we show that BRD4770-inducible FANCD2 and FANCI monoubiquitination and nuclear foci formation may be a consequence of inhibition of the PRC2/EZH2 chromatin-modifying complex. In addition, we show that inhibition of the class I and II histone deacetylases leads to attenuated FANCD2 and FANCI monoubiquitination and nuclear foci formation. Our studies establish that chromatin state is a major determinant of the activation of the FA pathway and suggest an important role for the PRC2/EZH2 complex in the regulation of this critical tumor suppressor pathway.

Fanconi anemia (FA) is an inherited disease characterized by bone marrow failure and increased cancer risk. FA is caused by mutation of any 1 of 22 genes, and the FA proteins function cooperatively to repair DNA interstrand cross-links (ICLs). A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which occurs within chromatin. How FANCD2 and FANCI are anchored to chromatin remains unknown. In this study, we identify and characterize a FANCD2 histone-binding domain (HBD) and embedded methyl-lysine-binding domain (MBD) and demonstrate binding specificity for H4K20me2. Disruption of the HBD/MBD compromises FANCD2 chromatin binding and nuclear focus formation and its ability to promote error-free DNA interstrand cross-link repair, leading to increased error-prone repair and genome instability. Our study functionally describes the first FA protein chromatin reader domain and establishes an important link between this human genetic disease and chromatin plasticity.

Fanconi anemia (FA) is a rare genetic disease characterized by increased risk for bone marrow failure and cancer. The FA proteins function together to repair damaged DNA. A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which occurs upon exposure to DNA damaging agents and during S-phase of the cell cycle. The regulatory mechanisms governing S-phase monoubiquitination, in particular, are poorly understood. In this study, we have identified a CDK regulatory phospho-site (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 S592 phosphorylation was detected by LC-MS/MS and by immunoblotting with a S592 phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination of FANCD2 during S-phase. Furthermore, FA-D2 ( FANCD2 -/- ) patient cells expressing S592 mutants display reduced proliferation under conditions of replication stress and increased mitotic aberrations, including micronuclei and multinucleated cells. Our findings describe a novel cell cycle-specific regulatory mechanism for the FANCD2 protein that promotes mitotic fidelity.

SWI/SNF has been shown to have important functions in hypoxia-mediated gene expression through roles of its catalytic and core subunits. Since SWI/SNF exists as three distinct assemblies, and usage of complex specific subunits of the complex can be expected to vary within a given cell under changing environmental conditions. It remains an open question as to the compositional makeup of SWI/SNF and the roles of individual complexes in gene expression and cell viability in a hypoxic environment. In our current study, we find that hypoxia regulates levels of unique subunits that define each complex. Protein levels of ARID2 and PBRM1, members of PBAF and BRD9, a member of ncBAF, are downregulated in hypoxic cells, while members of BAF complex are retained. Our studies further show that loss of ARID1A, ARID1B and DPF2, which are unique subunits of BAF, reduces induction of HIF target genes and ARID1A or DPF2 are important for cell survival during hypoxia. Collectively, our results provide evidence that levels of SWI/SNF forms are not static within cells, but can be dynamically altered as a response to environmental changes.

Fanconi anemia (FA) is a rare genetic disease characterized by increased risk for bone marrow failure and cancer. The FA proteins function together to repair damaged DNA. A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins under conditions of cellular stress and during S-phase of the cell cycle. The regulatory mechanisms governing S-phase monoubiquitination, in particular, are poorly understood. In this study, we have identified a CDK regulatory phospho-site (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 S592 phosphorylation was detected by LC-MS/MS and by immunoblotting with a S592 phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination of FANCD2 during S-phase. Furthermore, FA-D2 (FANCD2-/-) patient cells expressing S592 mutants display reduced proliferation under conditions of replication stress and increased mitotic aberrations, including micronuclei and multinucleated cells. Our findings describe a novel cell cycle-specific regulatory mechanism for the FANCD2 protein that promotes mitotic fidelity.Author SummaryFanconi anemia (FA) is a rare genetic disease characterized by high risk for bone marrow failure and cancer. FA has strong genetic and biochemical links to hereditary breast and ovarian cancer. The FA proteins function to repair DNA damage and to maintain genome stability. The FANCD2 protein functions at a critical stage of the FA pathway and its posttranslational modification is defective in >90% of FA patients. However, the function, and regulation of FANCD2, particularly under unperturbed cellular conditions, remains remarkably poorly characterized. In this study, we describe a novel mechanism of regulation of the FANCD2 protein during S-phase of the cell cycle. CDK-mediated phosphorylation of FANCD2 on S592 promotes the ubiquitination of FANCD2 during S-phase. Disruption of this phospho-regulatory mechanism results in compromised mitotic fidelity and an increase in mitotic chromosome instability.

ARID1A, a subunit of SWI/SNF, has been shown to play a major role in recruitment of the chromatin remodeler to enhancers for transcriptional regulation. Mutations inARID1A have been found in various cancers, many of which form solid tumors. Recent studies have revealed vulnerabilities in cells lacking ARID1A, specifically ARID1B, an ortholog and mutually exclusive subunit, in 2D cell culture. However, identification of vulnerabilities within SWI/SNF for loss of ARID1A in a multicellular tumor spheroid, that mimic in vivo condition within tumors, has not been explored. Here we show in the absence of ARID1A in a MTS model, ARID1B continues to be a vulnerability but we have identified PBRM1 as a new vulnerability within SWI/SNF. Levels of ARID1B and PBRM1 are elevated on loss of ARID1A. Further, reduction of ARID1B and PBRM1 protein levels, decreases cell survival and reduces induction of several hypoxia regulated genes in ARID1A deficient MTSs. Our studies have identified PBRM1 as a new vulnerability in ARID1a deficient cancers and which provides a new target for therapeutic strategies.