Interleukin-12 and TNFα signaling drives parallel evolution of independent leukemic clones in JAK2V617F mutant myeloproliferative neoplasms Free

Progression from chronic phase myeloproliferative neoplasm (MPN) to secondary acute myeloid leukemia (sAML) is a poorly understood, severe complication traditionally linked to additional mutations in the MPN driver clone in genes such as TET2 and TP53. However, the absence of the JAK2 driver mutation in sAML cases which transform from JAK2-mutant MPNs suggests alternative mechanisms. We confirmed with single-cell DNA sequencing of paired MPN and sAML patient samples that sAML driver mutations arise from non-JAK2-mutant clones in a portion of post-MPN sAML cases. We established a series of models mirroring this progression by utilizing mouse strains with inducible expression of Jak2^V617F (Vav-Cre;Jak2^V617F/+) and Tet2 heterozygous loss-of-function (Vav-Cre;Tet2^fl/+). Additionally, we established patient-derived xenografts (PDXs) to capture these dynamics by transplanting cord blood derived CD34+ cells engineered to possess TET2 mutations with CD34+ cells derived from healthy donor bone marrow (BM; control) or JAK2-mutant MPN patients. These models replicate the emergence of leukemic clones (Tet2) that outcompete the MPN driver clone (Jak2), particularly in hematopoietic progenitor (c-Kit+ Sca-1+ Lineage- “KSL”) cells.

We utilized these experimental systems to identify cell intrinsic and cell extrinsic mechanisms by which MPN cells support parallel clones. Bulk RNA sequencing of sorted KSL cells from murine chimeras revealed that in a Jak2-mutant environment, Tet2-mutant KSL cells were biased towards proliferation (Mki67) and myeloid differentiation (Elane and Mpo) at the relative expense of self-renewal (Hoxa10, Fgd5, Vldlr, Hlf, and Mecom) when compared to Tet2-mutant KSL cells from a WT environment. Interestingly, KSL cells isolated from these chimeras at earlier timepoints demonstrated higher levels of dysregulation in these targets indicating that the transcriptional dysregulation is at least partially driven by Jak2-mutant cells which are subsequently out-competed over time leading to a diminished signal. Over representation analysis identified IL-12-mediated signaling as the most dysregulated cancer-related pathway. Cytokine profiling revealed a positive correlation between IL-12 and TNFα levels and increasing TET2-mutant expansion in both human and murine chimeras. Intracellular flow cytometry revealed pSTAT4 (canonical readout of IL-12) was most elevated in Tet2-mutant BM cells isolated from a Jak2-mutant background compared to those from a WT background or from WT cells from either background.

To test the functional implications of TNFα on the observed competitive advantage of Tet2-mutant clones in a Jak2-mutant environment, Tet2^fl/+ mice were crossed with a TNF-receptor genetic deletion model (p55/p75 germline KO) to establish mice wherein Tet2-mutant cells lack receptors for TNFα. BM cells from either Jak2^V617F or WT mice were mixed with either Tet2^fl/+, TNFp55/75 KO, Tet2^fl/+/TNFp55/75 KO, or WT test BM and co-transplanted with WT support. Peripheral blood and BM analysis revealed the competitive advantage of Tet2-mutant cells in a Jak2-mutant environment was significantly blunted with genetic deletion of TNF receptors. To examine this finding in a more translationally relevant system, we administered murine biosimilars of TNFα and IL-12 monoclonal neutralizing antibodies (adalimumab/ustekinumab respectively) into our chimeric systems and the human agonist into our PDX system. In all cases, the inhibition of TNFα and IL-12 resulted in limited Tet2-mutant expansion in a Jak2-mutant background. qPCR analysis performed on sorted Tet2-mutant KSL cells isolated from control and treated cohorts, including those with TNF receptor deletion, revealed that targeting the IL-12/TNFα axis significantly reduced genetic dysregulation in proliferation and self-renewal pathways. This aligns with the reduced expansion of Tet2-mutant blood, BM, and progenitor cells observed in these cohorts. These data establish that JAK2-mutant MPN cells facilitate the parallel expansion of independent clones via an IL-12/TNFα axis which biasesTet2-mutant progenitors towards myeloid differentiation and increases proliferation at the expense of self-renewal. Genetic and pharmacological inhibition of IL-12 and TNFα resulted in both functional and molecular rescue of this phenotype. We aim to leverage these findings to enhance disease surveillance in MPN populations and identify candidates for potential interventional therapy.