TNFα facilitates clonal expansion of JAK2V617F positive cells in myeloproliferative neoplasms.

Proinflammatory cytokines such as TNFα are elevated in patients with myeloproliferative neoplasms (MPN), but their contribution to disease pathogenesis is unknown. Here we reveal a central role for TNFα in promoting clonal dominance of JAK2(V617F) expressing cells in MPN. We show that JAK2(V617F) kinase regulates TNFα expression in cell lines and primary MPN cells and TNFα expression is correlated with JAK2(V617F) allele burden. In clonogenic assays, normal controls show reduced colony formation in the presence of TNFα while colony formation by JAK2(V617F)-positive progenitor cells is resistant or stimulated by exposure to TNFα. Ectopic JAK2(V617F) expression confers TNFα resistance to normal murine progenitor cells and overcomes inherent TNFα hypersensitivity of Fanconi anemia complementation group C deficient progenitors. Lastly, absence of TNFα limits clonal expansion and attenuates disease in a murine model of JAK2(V617F)-positive MPN. Altogether our data are consistent with a model where JAK2(V617F) promotes clonal selection by conferring TNFα resistance to a preneoplastic TNFα sensitive cell, while simultaneously generating a TNFα-rich environment. Mutations that confer resistance to environmental stem cell stressors are a recognized mechanism of clonal selection and leukemogenesis in bone marrow failure syndromes and our data suggest that this mechanism is also critical to clonal selection in MPN.

CYT387, a novel JAK2 inhibitor, induces hematologic responses and normalizes inflammatory cytokines in murine myeloproliferative neoplasms.

Activating alleles of Janus kinase 2 (JAK2) such as JAK2(V617F) are central to the pathogenesis of myeloproliferative neoplasms (MPN), suggesting that small molecule inhibitors targeting JAK2 may be therapeutically useful. We have identified an aminopyrimidine derivative (CYT387), which inhibits JAK1, JAK2, and tyrosine kinase 2 (TYK2) at low nanomolar concentrations, with few additional targets. Between 0.5 and 1.5muM CYT387 caused growth suppression and apoptosis in JAK2-dependent hematopoietic cell lines, while nonhematopoietic cell lines were unaffected. In a murine MPN model, CYT387 normalized white cell counts, hematocrit, spleen size, and restored physiologic levels of inflammatory cytokines. Despite the hematologic responses and reduction of the JAK2(V617F) allele burden, JAK2(V617F) cells persisted and MPN recurred upon cessation of treatment, suggesting that JAK2 inhibitors may be unable to eliminate JAK2(V617F) cells, consistent with preliminary results from clinical trials of JAK2 inhibitors in myelofibrosis. While the clinical benefit of JAK2 inhibitors may be substantial, not the least due to reduction of inflammatory cytokines and symptomatic improvement, our data add to increasing evidence that kinase inhibitor monotherapy of malignant disease is not curative, suggesting a need for drug combinations to optimally target the malignant cells.

Absence of SKP2 expression attenuates BCR-ABL-induced myeloproliferative disease.

BCR-ABL is proposed to impair cell-cycle control by disabling p27, a tumor suppressor that inhibits cyclin-dependent kinases. We show that in cell lines p27 expression is inversely correlated with expression of SKP2, the F-box protein of SCF(SKP2) (SKP1/Cul1/F-box), the E3 ubiquitin ligase that promotes proteasomal degradation of p27. Inhibition of BCR-ABL kinase causes G(1) arrest, down-regulation of SKP2, and accumulation of p27. Ectopic expression of wild-type SKP2, but not a mutant unable to recognize p27, partially rescues cell-cycle progression. A similar regulation pattern is seen in cell lines transformed by FLT3-ITD, JAK2(V617F), and TEL-PDGFRbeta, suggesting that the SKP2/p27 conduit may be a universal target for leukemogenic tyrosine kinases. Mice that received transplants of BCR-ABL-infected SKP2(-/-) marrow developed a myeloproliferative syndrome but survival was significantly prolonged compared with recipients of BCR-ABL-expressing SKP2(+/+) marrow. SKP2(-/-) leukemic cells demonstrated higher levels of nuclear p27 than SKP2(+/+) counterparts, suggesting that the attenuation of leukemogenesis depends on increased p27 expression. Our data identify SKP2 as a crucial mediator of BCR-ABL-induced leukemogenesis and provide the first in vivo evidence that SKP2 promotes oncogenesis. Hence, stabilization of p27 by inhibiting its recognition by SCF(SKP2) may be therapeutically useful.

On-target restoration of a split T cell-engaging antibody for precision immunotherapy.

T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (VL) or variable heavy (VH) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies.

Characterization of murine JAK2V617F-positive myeloproliferative disease.

The JAK2(V617F) mutation is present in almost all patients with polycythemia vera (PV), large proportions of patients with essential thrombocythemia and idiopathic myelofibrosis, and less frequently in atypical myeloproliferative disorders (MPD). We show that transplantation of JAK2(V617F)-transduced bone marrow into BALB/c mice induces MPD reminiscent of human PV, characterized by erythrocytosis, granulocytosis, extramedullary hematopoiesis, and bone marrow fibrosis, but not thrombocytosis. Fluorescence-activated cell sorting of bone marrow and spleen showed proportional expansion of common myeloid progenitors, granulocyte-monocyte and megakaryocyte-erythrocyte progenitors. Megakaryocyte and late erythroid progenitors were dramatically increased, with only modest expansion of early erythroid progenitors. Erythropoietin (Epo) receptor expression was reduced on early, but normal on late erythroblasts. Serum levels of Epo and granulocyte colony-stimulating factor, but not granulocyte macrophage colony-stimulating factor, were reduced, whereas tumor necrosis factor-alpha was increased, possibly exerting a negative effect on JAK2(V617F)-negative hematopoiesis. These data suggest that erythrocytosis and granulocytosis in JAK2(V617F) mice are the net result of a complex interplay between cell intrinsic and extrinsic factors. There were no thromboembolic events and no animals succumbed to their disease, implicating additional factors in the manifestation of human disease. The disease was not transplantable and prolonged observation showed normalization of blood counts in most JAK2(V617F) mice, suggesting that the mutation may not confer self-renewal capacity.