Abstract
Polycythemia vera (PV) is a clonal disorder arising from a single stem cell while normal stem cells are present in the marrow but are suppressed by the PV clone by an unknown mechanism. Pegylated interferon alfa-2a (PegInfa), a better-tolerated form of Infa induces clinical remission, reduces JAK2V617F allelic burden, and may convert clonal to polyclonal hematopoiesis (EL Liu, Blood 2003). Tumor necrosis factor-α (TNFa) levels are increased in patients with myeloproliferative neoplasms, including PV (Fleischman, Blood 2011). We previously reported that transcripts of TNFα mRNA are higher in CD34+ cells compared to more differentiated cells. TNFa is markedly reduced in those PegInfa-treated patients with decreased JAK2V617F allelic burden and/or return of polyclonal hematopoiesis (detected in females by X-chromosome allelic usage ratio), while no such decrease was seen in PV with hydroxyurea-induced normalization of elevated hematocrit/platelets/leukocytes (Swierczek, ASH 2012). To directly interrogate the role of TNFα in inducing suppression of normal hematopoiesis, we used a TNFα blocking antibody (adalimumab) and examined its role on PV erythropoiesis using a 3-week liquid culture system characterized by synchronized differentiation of expanding erythroid progenitors (Bruchova, Exp Hemat, 2007).
In vitro expanded PV erythroid progenitors were grown with or without adalimumab or PegInfa. We evaluated apoptosis, proliferation and differentiation at different stages of erythroid maturation and correlated these parameters with TNFα transcripts, JAK2V617F allelic burden and, in females, clonality. Although the initial mononuclear cells represented a heterogeneous population, the expansion process favors erythroid progenitors and results in their synchronized differentiation. The JAK2V617F allelic burden increased concomitant with erythroid expansion and reached its peak at day 11 (when the majority of cells are proerythroblasts and basophilic erythroblasts), indicating a preferential expansion of erythroid progenitors, and then declined progressively. The addition of adalimumab markedly reduced TNFα mRNA and JAK2V617F allelic burden compared to controls. We previously reported that in this in vitro liquid expansion system, PV erythroid progenitors exhibit accelerated differentiation at days 7-14 and increased proliferation at days 9-14, with a larger S-phase population (40%) than controls (20%) at day 11 (Bruchova Exp Hemat, 2007). Compared to controls, adalimumab increased proliferation and delayed differentiation at early stages of PV erythropoiesis, with the proportion of apoptotic cells consistently decreased compared to erythroid cells expanded without adalimumab. Furthermore, X-chromosome-based clonality assays revealed preferential expansion of normal progenitors in 1 informative female patient. We also measured the impact of TNFα inhibition with adalimumab on burst-forming units-erythroid (BFU-E) colonies from PV patients cultured ex vivo. As expected, JAK2WT, JAK2WT/V617F and JAK2V617F BFU-E colonies were detected in cultures performed in the absence of adalimumab, while the addition of adalimumab preferentially abrogated JAK2V617F homozygous BFU-Es.
In analogous experiments, PegInfa markedly decreased TNFα mRNA and JAK2V617F allelic burden, and decreased differentiation, but unlike adalimumab, it decreased proliferation and had no demonstrable effect on apoptosis. Ongoing studies are being performed to correlate the TNFα mRNA expression changes seen with adalimumab treatment in erythroid progenitors with differences in TNFα protein levels using intracellular cytokine staining and flow cytometry.
These data suggest that blocking TNFα can suppress the JAK2V617F clone, rescue normal dormant hematopoiesis, and provide a foundation for a combined PV therapy using TNFα blockers with either PegInfa or JAK2 inhibitors.
Disclosures
Deininger: BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMS, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.
Using the Minor Variant Finder software to identify and quantify the allelic burden level of somatic mutations in oncohematologic diseases