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330 Jak2V617F Reversible Activation Shows an Essential Requirement for Jak2V617F in Myeloproliferative Neoplasms (MPNs)

Program: Oral and Poster Abstracts
Type: Oral
Session: 631. Myeloproliferative Syndromes and Chronic Myeloid Leukemia: Basic and Translational: Identification of Novel Therapeutic Targets in MPN and CML
Hematology Disease Topics & Pathways:
Research, Translational Research, MPN, Chronic Myeloid Malignancies, Diseases, Myeloid Malignancies
Saturday, December 10, 2022: 5:15 PM

Andrew Dunbar, MD1, Robert L. Bowman, PhD2, Young Park, MS3*, Franco Izzo, PhD4, Robert M. Myers5, Abdul Karzai, BS3*, Won Jun Kim3*, Inés Fernández Maestre3*, Michael R. Waarts6*, Abbas Nazir3*, Wenbin Xiao, MD, PhD7, Max Brodsky, MD8, Mirko Farina, MD9*, Louise Cai6*, Sheng F Cai, MD, PhD10, Benjamin Wang11*, Wenbin An6*, Julie Yang, PhD12*, Shoron Mowla6*, Shira E. Eisman6*, Tanmay Mishra6*, Remie Houston3*, Emily Guzzardi, BA3*, Anthony R. Martinez Benitez, BA3*, Aaron D Viny, MD, MS13, Richard Koche, PhD12*, Dan A. Landau, MD, PhD14 and Ross L. Levine, MD6

1Department of Medicine and Center for Hematologic Malignancies, Memorial Sloan Kettering Cancer Center, New York, NY
2Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA
3Center for Hematologic Malignancies, Memorial Sloan Kettering Cancer Center, New York, NY
4Meyer Cancer Center & Institute of Computational Biomedicine, Weill Cornell Medicine, New York City, NY
5Tri-Institutional MD-PhD Program, Weill Cornell Medicine, Rockefeller University, Memorial Sloan Kettering Cancer Center, New York, NY
6Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY
7Department of Pathology and Laboratory Medicine, Hematopathology Service, Memorial Sloan Kettering Cancer Center, New York, NY
8Department of Medicine, Johns Hopkins University, Baltimore, MD
9Unit of Blood Diseases and Stem Cell Transplantation, ASST Spedali Civili, Department of Clinical and Experimental Sciences, University of Brescia, Brescia, Pavia, Italy
10Department of Medicine, Leukemia Service, Memorial Sloan Kettering Cancer Center, New York, NY
11Center for Hematologic Malignancies, Memorial Sloan Kettering Cancer Center, New York
12Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY
13Columbia Stem Cell Initiative, Columbia University, New York, NY
14Meyer Cancer Center & Institute of Computational Biomedicine, Weill Cornell Medicine, New York, NY

JAK inhibitors (JAKi) improve symptoms and outcomes in MPNs; however, mutant allele burden does not substantively change with chronic therapy. Alternative MAPK signaling and/or cooperating mutations are thought to contribute to MPN cell persistence; however, we hypothesized current JAKi fail to effectively abrogate mutant JAK2 signaling. To that end, we developed a conditionally inducible mouse model allowing for sequential activation, then deletion, of Jak2VF from its endogenous locus using a Dre-rox/Cre-lox (Jak2RL) dual recombinase system (Figure 1A).

Dre mRNA electroporation of lineage-negative UbcCreER-Jak2RL bone marrow (BM) was used to induce Jak2VF ex vivo, followed by transplant into lethally-irradiated recipient mice in competition with Cd45.1 BM. Transplanted mice developed a highly penetrant MPN characterized by leukocytosis, polycythemia, hepatosplenomegaly, and BM megakaryocytic hyperplasia. Tamoxifen administration 12 weeks post-transplant to delete Jak2VF resulted in abrupt normalization of blood cell parameters, spleen weights, and serum pro-inflammatory cytokines, with a marked prolongation in overall survival (OS) compared to MPN control mice. Reduced peripheral blood (PB) and BM Cd45.2 mutant fraction was also observed, including within hematopoietic stem/progenitor cell (HSPC) compartments and to a greater degree than ruxolitinib (RUX) therapy. Notably, treatment with CHZ868, a type II JAKi, showed improvements in regard to hematocrit and spleen weight reduction on par with deletion, as well as significant reductions in BM HSPC mutant fractions, albeit to a lesser degree than with Jak2VF reversion (Figure 1B). Transplant of Jak2VF deleted BM failed to form phenotypic disease in secondary recipient mice consistent with loss of disease-propagating MPN stem cells. RNA-Seq on sorted LSKs following Jak2VF deletion showed loss of STAT5, MAPK, and MTORC1 target gene expression, as well as negative enrichment in TGFb and TNFa/NFkB pathways consistent with reduced pro-inflammatory and proliferative output. The alterations in MAPK signaling were supported by Ybx1 RT-PCR of BM cKit+ cells and BM pERK IHC, both of which were increased with RUX but potently suppressed with deletion suggesting improved JAK2 targeting can overcome MAPK-mediated persistence.

The initial proximity of Jak2RL lox sites grants Cre-mediated recombination prior to Dre-mediated Jak2VF knock-in allowing for assessment of Jak2VF oncogenic dependency alone or in the context of co-occurring mutation. Given the known effects of Tet2 loss on enhanced MPN HSC fitness, we evaluated Tet2 loss on sustaining MPN with Jak2VF reversion. UbcCreER-Jak2RL/Tet2f/f mice were pulsed with TAM to knock-out Tet2 followed by sacrifice and Dre Jak2RL knock-in 6 weeks later. Mice transplanted with UbcCreER-Jak2VF/Tet2-/- cells exhibited enhanced leukocytosis, spleen weights and PB/BM chimerism compared to Jak2VF mice; however, Jak2VF reversion resulted in similar reductions in MPN disease parameters, including Cd45.2 fraction, suggesting Tet2 loss does not dramatically alter dependence on mutant JAK/STAT signaling. Notably, while Jak2VF/Tet2-/- mutant cells displayed increased serial replating capacity, this was abrogated with Jak2VF reversion suggesting despite the enhanced fitness of Tet2­-/- HSCs, Jak2VF creates a unique dependency that renders double-mutant cells susceptible to eradication with Jak2VF reversion.

In sum, Jak2VF deletion abrogates MPN features, extends OS, and reduces mutant cell fraction with depletion of disease-sustaining MPN HSCs, either alone or in the setting of cooperating Tet2 loss. These data suggest JAK2VF mutant-selective inhibition offers greater therapeutic potential than current JAKi and a potential curative option for the treatment of MPN.

Disclosures: Bowman: Mission Bio: Honoraria, Speakers Bureau. Cai: Imago Biosciences: Consultancy, Current equity holder in publicly-traded company. Viny: Arima Genomics: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Nooma Bio: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Landau: Abbvie and Illumina: Consultancy; Mission Bio and C2i Genomics: Other: Scientific Advisory Board; BMS, 10X Genomics, and Illumina: Research Funding. Levine: Imago, Mission Bio, Bakx, Zentalis, Ajax, Auron, Prelude, C4 Therapeutics and Isoplexis: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Ajax, Abbvie, Constellation, Zenalis, Celgene, Roche, and Prelude: Other: research support; Syndax, Incyte, Janssen, Astellas, Morphosys and Novartis: Consultancy; Astra Zeneca and Kura: Other: honoraria for invited lectures ; Gilead and Novartis: Other: Grant reviews; Qiagen: Other: supervisory board member.

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