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2712 TET2 and TP53 Mutations Cooperate in Leukemia Development and Modulate the Response to Inflammation

Program: Oral and Poster Abstracts
Session: 602. Myeloid Oncogenesis: Basic: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Pablo Sanchez Vela, MD1*, Brianna Kelly2*, Claudia Brady3*, Jonathan Foox4*, Jacob Glass, MD, PhD5, Ross L Levine, MD1 and Alan Shih, MD, PhD2

1Memorial Sloan Kettering Cancer Center, New York, NY
2Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY
3Icahn School of Medicine at Mount Sinai, New York, NY
4Weill Cornell Medicine, New York, NY
5Leukemia Service, Department of Medicine, Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY

Introduction

TET2, a regulator of DNA methylation, is amongst the most commonly mutated genes in acute myeloid leukemia (AML) and is linked to inflammation response. In AML TP53 can be affected through mutation, deletion, and dysregulation of modifiers such as PPM1D and MDM2. These mutations co-occur in select cases of acute leukemias. We assessed whether concomitant TET2 and TP53 mutations would promote hematopoietic transformation and alter stem-cell fitness and response to cell extrinsic factors.

Methods

We genetically combined deletion mutation alleles of Tet2 and Tp53 in a mouse model with Mx1-Cre and Scl-CreERT2 to allow for somatic excision of Tet2 with administration of PIPC or Tamoxifen. Bone marrow transplant (BMT) experiments with wild-type, single mutant Tet2 or Tp53, and double mutant bone marrow were performed with peripheral blood counts, cytokine ELISA, and immune-phenotyping. A conditional Jak1 allele was evaluated by additional crosses. In vivo inflammation was induced LPS. Hematopoietic stem-progenitor cells (HSPCs) from leukemic disease mice were profiled by RNA-seq and bisulfite sequencing. Colony forming assay was performed using methylcellulose. CRISPR KO of NLRP1 lines were generated by lentivirus transduction.

Results

Tet2 Tp53 mutant mice developed myeloproliferative neoplasm with myeloid biased white blood counts (mean, Mac+Gr+ 25% vs 15% WT) and splenomegaly (mean, 310 mg vs 81 mg WT). In BMTs, Tet2 Tp53 mutant-transplanted mice developed more aggressive disease (median survival 150 days vs not reached for WT and single mutant). Furthermore, double mutant cells had competitive growth advantage over wild-type and single mutant cells. Starting from 50% of transplanted cells, double mutant cells expanded to 89% vs WT, 87% vs Tp53-/-, and 66% vs Tet2-/-. In addition, in Tet2-/- Tp53-/- mice immature B-lymphoctyes were more often observed (B-220dim, >5% in ~30% of mice), and transplanted mice developed both myeloid and lymphoid (B-cell) leukemia.

RNA-seq showed enriched inflammatory gene signatures associated with double mutant leukemic HSPCs, including IFNγ activation (adj p <.001). Methylation analysis did not find a correlation between methylation and expression. Serum cytokine levels in Tet2-/- Tp53-/- disease mice were elevated compared to wild-type, TNFα (mean, 92.7 pg/mL vs 29.7 pg/mL) and IFNγ (mean, 12.9 pg/mL vs 5.4 pg/mL). Next, we subjected HSPCs to colony forming assays with cytokines. IFNγ reduced colony formation potential, but Tet2 Tp53 mutant cells were able to sustain colony formation for further re-platings (CFU replating 4-6 vs WT 2-3). In addition transplanted Tet2-/- Tp53-/- cells further outcompeted wild-type bone marrow with LPS treatment in vivo (WT declined by 40% with vehicle but 70% with LPS).

Amongst inflammation-associated genes Nlrp1 expression is decreased in Tp53-/- cells, and we found TP53 to be a regulator of NLRP1. By qPCR induction of NLRP11 was similar to MDM2 when cells were treated with the p53 activator AMG232 (MDM2 inhibitor). NLRP1 is a component of the inflammasome that activates cytokine release and pyroptotic cell death. It is also a sensor of ribotoxic stress, i.e. ribosome stalling due to cellular stress and chemotherapy such as daunorubicin. NLRP1-KO in OCI-AML3 leukemia cells leads to dampening of this pathway as measured by reduced caspase1 activity and cleavage of gasdermin D. Thus, TP53 mutations may lead to increased ability to tolerate inflammatory stress by limiting NLRP1-related activation.

To further understand Tet2-Trp53 double mutant cells and inflammatory stress response, we crossed this leukemia model to SclCreERT2 and Jak1 floxed mice (a mediator of IFNγ signaling) and performed competitive BMT. Whereas the chimerism of double mutant myeloid cells was extremely high (mean, 93%), the triple mutant cells (Tet2-Trp53-Jak1 KO) was more comparable to that of WT cells (mean, 40%). Thus, IFNγ-Jak1-mediated inflammation is required for enhanced myelopoiesis.

Conclusion

Co-occurring TET2 and TP53 mutations cooperate to promote leukemia progression. Mutations provide growth advantage to stem cells at steady state and also with inflammatory stress, including IFNγ induced senescence. TP53 regulates NLRP1, a component of this inflammatory stress response, contributing to greater HSPC tolerance of this stress pathway and thus contributes to the cooperativity with pro-inflammatory TET2-mutations.

Disclosures: Levine: Mana: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bridge Medicines: Consultancy; Jubilant: Membership on an entity's Board of Directors or advisory committees; Kurome: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Anovia: Consultancy; Prelude Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bridge Bio: Consultancy; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Epiphanes: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; Scorpion: Membership on an entity's Board of Directors or advisory committees; Bakx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy; Imago: Consultancy; Zentalis: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees.

*signifies non-member of ASH