Leveraging clonal competition between chip and malignant clones as a novel therapeutic strategy in hematologic malignancy Free

Hematopoiesis is a polyclonal process in which hematopoietic stem cells (HSCs) are in constant competition within the bone marrow niche. With aging and chronic inflammatory stress, HSC bearing somatic mutations that confer a fitness advantage may accumulate at the expense of their less fit competitors, giving rise to clonal hematopoiesis of indeterminate potential (CHIP). Although CHIP-associated clones, such as those with TET2 or DNMT3A mutations, are typically subclinical, they reflect enhanced stem cell fitness and persistence under stress. In contrast, malignant clones often overtake and disrupt normal hematopoiesis, leading to disease. We hypothesized that the selective advantage of CHIP clones could be harnessed to suppress the expansion of malignant clones through intrinsic clonal competition. To test whether CHIP HSCs can outcompete malignant clones, we evaluated the ability of Tet2⁻/⁻ cells to suppress disease development in two mouse models of hematologic malignancy: MPL^W515L as a model of aggressive disease, and Jak2^V617Fas a model of more chronic clonal expansion.

First, we used the well-established transduction-transplantation model of MPL^W515L as a model system for aggressive hematologic malignancy. This mouse model results in leukocytosis, thrombocytosis, splenomegaly, reticulin fibrosis in bone marrow and spleen, and leads to death by one-month post-transplant. Whole bone marrow from wild-type C57B/6 (CD45.1) mice was transduced with Green Fluorescent Protein (GFP) tagged retrovirus to induce expression of hMPL^W515L. We transduced bone marrow with low viral multiplicity of infection (MOI) resulting in approximately 1-5% GFP⁺ cells to extend survival beyond the expected 3-4 weeks post-transplant. The transduced bone marrow was mixed with unmanipulated whole bone marrow from either WT or Tet2^-/- (both in CD45.1/2) to result in a 90%/10% mixture and transplanted into lethally irradiated CD45.2 mice. This design mimicked the emergence of an aggressive clone within a background of CHIP. Peripheral blood counts, %GFP⁺ cells, and chimerism was followed biweekly. Early lethality occurred in ~10% of mice in both groups due to rapid disease onset. Among surviving mice, Tet2⁻/⁻ competitors significantly reduced hMPL^W515L burden, with 23% GFP+ cells compared to 55% in wild-type competitors at day 105 (p<0.01). At five months, Tet2⁻/⁻ competitors maintained consistently lower GFP+ cell frequencies across hematopoietic compartments, including LKS stem/progenitor cells. Mice with Tet2⁻/⁻ competitors had reduced spleen and liver weights and preserved tissue architecture, in contrast to the disrupted histology seen with wild-type competitors.

To assess clonal competition in a more chronic disease context, we used a knock-in model of Jak2^V617F. CD45.2 mice were first reconstituted with 500,000 CD45.1 Tet2⁻/⁻ or wild-type bone marrow cells to create mice with CHIP. After two months, mice were sublethally irradiated (100 cGy) and transplanted with 2 million CD45.1/2 Jak2^V617F whole bone marrow cells. Expansion of Jak2V617F cells was significantly lower in the Tet2⁻/⁻ group (~5%) compared to WT (~45%, p<0.05). In granulocytes, Jak2^V617F clonal contribution reached ~60% in WT competitors vs. ~10% in the Tet2⁻/⁻ group (p<0.01). Wild-type recipients developed features consistent with polycythemia vera, including elevated red blood cell counts, whereas mice containing Tet2⁻/⁻ competitors had normal red blood cell counts. At sacrifice mice with WT competitors exhibited splenomegaly, whereas there was a significant reduction of splenic size in Tet2⁻/⁻ group (p<0.05). As observed in the peripheral blood, there was also a significant reduction of malignant VF expansion in the bone marrow of mice with Tet2⁻/⁻competitors compared to WT competitors.

Together, these findings provide proof of principle that HSPCs with enhanced competitive fitness and low malignant potential, such as Tet2⁻/⁻ cells, can suppress malignant clonal expansion in both aggressive and chronic models of myeloid disease. This approach represents a shift from conventional therapeutic strategies by focusing on restoring clonal balance rather than directly targeting malignant cells. Clonal competition may serve as a novel framework for controlling disease progression and stabilizing hematopoiesis in early-stage or high-risk hematologic malignancies.