JAK2V617F Mutant MPN Cells Support Parallel Evolution of Independent Leukemic Clones

Myeloproliferative neoplasms (MPNs) are clonal, hematological diseases driven by somatic mutations, predominantly JAK2^V617F. Progression to secondary acute myeloid leukemia (sAML) is coupled with a dismal prognosis and is one of the most feared complications for MPN patients. Transformation of MPN to sAML is traditionally thought to be driven by additional mutations in the MPN clone in genes such as TET2 and TP53. However, the JAK2^V617F driver mutation is occasionally absent in sAML which transforms from antecedent JAK2-mutant MPNs – a trajectory most frequently observed in polycythemia vera (PV) patients. Proposed explanations include somatic reversion and loss of heterozygosity, but an underexplored mechanism is that sAML originates from independent clones evolving in parallel to the MPN. Utilizing single-cell DNA sequencing of paired MPN and sAML patient samples, we show that sAML driver mutations arise from non-JAK2-mutant clones in a significant number of post-MPN sAML cases.

To study these clonal interactions, we utilized mouse models with inducible expression of Jak2^V617F (Vav-Cre;Jak2^V617F/+), Tet2 heterozygous loss-of-function (Vav-Cre;Tet2^fl/+), and Tp53 heterozygous knock-in (Tp53^R172H/+). Bone marrow (BM) cells from either Jak2^V617F or WT mice (2.5M cells; CD45.2) were mixed with either Tet2^fl/+, Tp53^R172H/+, or WT test BM (500K cells; CD45.1/2-GFP) and transplanted into lethally irradiated recipients (CD45.1) to produce a starting fraction of test BM of approximately 17%. 16-weeks post-transplant, Jak2^V617F-mutant BM significantly supported the expansion of both Tet2 (GFP%= 77 +/-1.9%) and Tp53 (GFP%= 64 +/-6.2%) mutant clones in the peripheral blood (PB) compared to WT host BM (Tet2 GFP%= 28 +/-1.4% and Tp53 GFP%= 25 +/-1.7%). BM analysis revealed the competitive advantage of Tet2-mutant cells was most pronounced in hematopoietic progenitor (c-Kit+ Sca-1+ Lineage- “KSL”) cells rather than long-term hematopoietic stem cells (KSL CD48- CD150+). A titration experiment determined that a Jak2-mutant BM cell burden of 35% was the threshold needed to support significant expansion of Tet2-mutant cells, a variant allele fraction (VAF) that did not support expansion of parallel Tp53^R172H/+ or WT cells.

To explore these dynamics in primary human cells, we engineered TET2^1216* and TP53^R248Q mutations (selected for highest prevalence in hematological cancers) into cord blood (CB)-derived human hematopoietic stem/progenitor cells (CD34+) using CRISPR/Cas9 editing. To establish patient-derived xenografts (PDXs), nucleofected CB-derived CD34+ cells (20K cells) were intratibially co-transplanted into immunodeficient mice with CD34+ cells derived from healthy human BM (control) or JAK2-mutant myelofibrosis (MF;n=5) or JAK2-mutant PV (n=5) patients (100K cells). TET2 and TP53 mutant allele burden was quantified by sequencing 18-weeks post-transplant. MF and PV host mice supported growth of TET2 and TP53 mutant clones significantly more than control healthy donor BM. Remarkably, all cohorts established from PV patients exhibited the highest expansion (up to 40x starting VAF) of independent pathogenic clones, particularly for TET2-mutant cells.

To identify mechanisms by which MPN cells support parallel clones, we performed cytokine profiling and found a positive correlation between IL-12 and TNFα serum levels with increasing TET2-mutant VAF in both human and mouse chimera models. Notably, TNFα has been reported to correlate with TET2-mutant clonal hematopoiesis and IL-12 is a known TNFα stimulator that is elevated in PV patients compared to other MPN subtypes. We hypothesized that IL-12 secreted by PV cells induces TNFα production resulting in an environment conditioned to favor expansion of TET2-mutant cells. Preliminary genetic and pharmacological studies suggest that neutralization of IL-12 decreases global TNFα and mitigates TET2-mutant clonal expansion in a Jak2-mutant background.

These data establish that JAK2^V617F-mutant MPN cells support parallel expansion of independent clones as one trajectory of evolution to sAML, particularly for PV patients. This mechanism is at least partly mediated through an IL-12/TNFα axis, which is amenable to therapeutic targeting. Our goal is to leverage these insights to improve disease surveillance and identify candidates for interventional therapy to mitigate leukemic clone emergence in MPN patients.