New insights into TCR ß-selection.

T cell receptor (TCR) ß-selection (herein referred to as ß-selection) is a pivotal checkpoint in mammalian T cell development when immature CD4-CD8- T-cells (thymocytes) express pre-TCR following successful Tcrb gene rearrangement. At this stage, αß T cell lineage commitment and allelic exclusion to restrict one ß-chain per cell take place and thymocytes undergo a proliferative burst. ß-selection is known to be crucially dependent upon synchronized Notch and pre-TCR signaling; however, other necessary inputs have been identified over the past decade, expanding our knowledge and understanding of the ß-selection process. In this review, we discuss recent mechanistic findings that have enabled a more detailed decoding of the molecular dynamics of the ß-selection checkpoint and have helped to elucidate its role in early T cell development.

Hmga2 promotes the development of myelofibrosis in Jak2V617F knockin mice by enhancing TGF-ß1 and Cxcl12 pathways.

Myelofibrosis (MF) is a devastating blood disorder. The JAK2V617F mutation has been detected in ∼50% cases of MF. Elevated expression of high-mobility group AT hook 2 (HMGA2) has also been frequently observed in patients with MF. Interestingly, upregulation of HMGA2 expression has been found in association with the JAK2V617F mutation in significant cases of MF. However, the contribution of HMGA2 in the pathogenesis of MF remains elusive. To determine the effects of concurrent expression of HMGA2 and JAK2V617F mutation in hematopoiesis, we transduced bone marrow cells from Jak2V617F knockin mice with lentivirus expressing Hmga2 and performed bone marrow transplantation. Expression of Hmga2 enhanced megakaryopoiesis, increased extramedullary hematopoiesis, and accelerated the development of MF in mice expressing Jak2V617F Mechanistically, the data show that expression of Hmga2 enhances the activation of transforming growth factor-ß1 (TGF-ß1) and Cxcl12 pathways in mice expressing Jak2V617F In addition, expression of Hmga2 causes upregulation of Fzd2, Ifi27l2a, and TGF-ß receptor 2. Forced expression of Cxcl12, Fzd2, or Ifi27l2a increases megakaryocytic differentiation and proliferation in the bone marrow of Jak2V617F mice, whereas TGF-ß1 or Cxcl12 stimulation induces collagen deposition in the bone marrow mesenchymal stromal cells. Together, these findings demonstrate that expression of Hmga2 cooperates with Jak2V617F in the pathogenesis of MF.

Epigenetic regulation of T cell development.

Epigenetic regulators are pivotal factors that influence and control T cell development. Recent findings continue to reveal additional elements of epigenetic modifications that play significant and crucial roles at different stages of T cell development. Through gaining a better understanding of the various epigenetic factors that influence the formation and survival of maturing T cells, new therapies can potentially be developed to combat diseases caused by dysregulated epigenetic chromatin modifications. In this review, we summarize the recent studies which shed light on the epigenetic regulation of T cell development especially at the critical stage of ß-selection.

SRSF2 mutation reduces polycythemia and impairs hematopoietic progenitor functions in JAK2V617F-driven myeloproliferative neoplasm.

SRSF2 mutations are found in association with JAK2V617F in myeloproliferative neoplasms (MPN), most frequently in myelofibrosis (MF). However, the contribution of SRSF2 mutation in JAK2V617F-driven MPN remains elusive. To investigate the consequences of SRSF2P95H and JAK2V617F mutations in MPN, we generated Cre-inducible Srsf2P95H/+Jak2V617F/+ knock-in mice. We show that co-expression of Srsf2P95H mutant reduced red blood cell, neutrophil, and platelet counts, attenuated splenomegaly but did not induce bone marrow fibrosis in Jak2V617F/+ mice. Furthermore, co-expression of Srsf2P95H diminished the competitiveness of Jak2V617F mutant hematopoietic stem/progenitor cells. We found that Srsf2P95H mutant reduced the TGF-ß levels but increased the expression of S100A8 and S100A9 in Jak2V617F/+ mice. Furthermore, enforced expression of S100A9 in Jak2V617F/+ mice bone marrow significantly reduced the red blood cell, hemoglobin, and hematocrit levels. Overall, these data suggest that concurrent expression of Srsf2P95H and Jak2V617F mutants reduces erythropoiesis but does not promote the development of bone marrow fibrosis in mice.

GRB2 promotes thymocyte positive selection by facilitating THEMIS-mediated inactivation of SHP1.

The T-lineage restricted protein THEMIS has been shown to play a critical role in T cell development. THEMIS, via its distinctive CABIT domains, inhibits the catalytic activity of the tyrosine phosphatase SHP1 (PTPN6). SHP1 and THEMIS bind to the ubiquitous cytosolic adapter GRB2, and the purported formation of a tri-molecular THEMIS-GRB2-SHP1 complex facilitates inactivation of SHP1 by THEMIS. The importance of this function of GRB2 among its numerous documented activities is unclear as GRB2 binds to multiple proteins and participates in several signaling responses in thymocytes. Here, we show that similar to Themis-/- thymocytes, the primary molecular defect in GRB2-deficient thymocytes is increased catalytically active SHP1 and the developmental block in GRB2-deficient thymocytes is alleviated by deletion or inhibition of SHP1 and is exacerbated by SHP1 overexpression. Thus, the principal role of GRB2 during T cell development is to promote THEMIS-mediated inactivation of SHP1 thereby enhancing the sensitivity of TCR signaling in CD4+CD8+ thymocytes to low affinity positively selecting self-ligands.

THEMIS increases TCR signaling in CD4+CD8+ thymocytes by inhibiting the activity of the tyrosine phosphatase SHP1.

The T cell lineage-restricted protein THEMIS plays a critical role in T cell development at the positive selection stage. In the SHP1 activation model, THEMIS is proposed to enhance the activity of the tyrosine phosphatase SHP1 (encoded by Ptpn6), thereby dampening T cell antigen receptor (TCR) signaling and preventing the inappropriate negative selection of CD4+CD8+ thymocytes by positively selecting ligands. In contrast, in the SHP1 inhibition model, THEMIS is proposed to suppress SHP1 activity, rendering CD4+CD8+ thymocytes more sensitive to TCR signaling initiated by low-affinity ligands to promote positive selection. We sought to resolve the controversy regarding the molecular function of THEMIS. We found that the defect in positive selection in Themis-/- thymocytes was ameliorated by pharmacologic inhibition of SHP1 or by deletion of Ptpn6 and was exacerbated by SHP1 overexpression. Moreover, overexpression of SHP1 phenocopied the Themis-/- developmental defect, whereas deletion of Ptpn6, Ptpn11 (encoding SHP2), or both did not result in a phenotype resembling that of Themis deficiency. Last, we found that thymocyte negative selection was not enhanced but was instead impaired in the absence of THEMIS. Together, these results provide evidence favoring the SHP1 inhibition model, supporting a mechanism whereby THEMIS functions to enhance the sensitivity of CD4+CD8+ thymocytes to TCR signaling, enabling positive selection by low-affinity, self-ligand-TCR interactions.

Interleukin-1 contributes to clonal expansion and progression of bone marrow fibrosis in JAK2V617F-induced myeloproliferative neoplasm.

Chronic inflammation is frequently associated with myeloproliferative neoplasms (MPN), but the role of inflammation in the pathogenesis of MPN remains unclear. Expression of the proinflammatory cytokine interleukin-1 (IL-1) is elevated in patients with MPN as well as in Jak2V617F knock-in mice. Here, we show that genetic deletion of IL-1 receptor 1 (IL-1R1) normalizes peripheral blood counts, reduces splenomegaly and ameliorates bone marrow fibrosis in homozygous Jak2V617F mouse model of myelofibrosis. Deletion of IL-1R1 also significantly reduces Jak2V617F mutant hematopoietic stem/progenitor cells. Exogenous administration of IL-1ß enhances myeloid cell expansion and accelerates the development of bone marrow fibrosis in heterozygous Jak2V617F mice. Furthermore, treatment with anti-IL-1R1 antibodies significantly reduces leukocytosis and splenomegaly, and ameliorates bone marrow fibrosis in homozygous Jak2V617F mice. Collectively, these results suggest that IL-1 signaling plays a pathogenic role in MPN disease progression, and targeting of IL-1R1 could be a useful strategy for the treatment of myelofibrosis.

Genetic ablation of Pim1 or pharmacologic inhibition with TP-3654 ameliorates myelofibrosis in murine models.

Myelofibrosis (MF) is the deadliest form of myeloproliferative neoplasm (MPN). The JAK inhibitor Ruxolitinib can reduce constitutional symptoms but it does not substantially improve bone marrow fibrosis. Pim1 expression is significantly elevated in MPN/MF hematopoietic progenitors. Here, we show that genetic ablation of Pim1 blocked the development of myelofibrosis induced by Jak2V617F and MPLW515L. Pharmacologic inhibition of Pim1 with a second-generation Pim kinase inhibitor TP-3654 significantly reduced leukocytosis and splenomegaly, and attenuated bone marrow fibrosis in Jak2V617F and MPLW515L mouse models of MF. Combined treatment of TP-3654 and Ruxolitinib resulted in greater reduction of spleen size, normalization of blood leukocyte counts and abrogation of bone marrow fibrosis in murine models of MF. TP-3654 treatment also preferentially inhibited Jak2V617F mutant hematopoietic progenitors in mice. Mechanistically, we show that TP-3654 treatment significantly inhibits mTORC1, MYC and TGF-ß signaling in Jak2V617F mutant hematopoietic cells and diminishes the expression of fibrotic markers in the bone marrow. Collectively, our results suggest that Pim1 plays an important role in the pathogenesis of MF, and inhibition of Pim1 with TP-3654 might be useful for treatment of MF.

New Insights into Epigenetic Regulation of T Cell Differentiation.

Immature CD4- CD8- thymocytes progress through several developmental steps in the thymus, ultimately emerging as mature CD4+ (helper) or CD8+ (cytotoxic) T cells. Activation of naïve CD4+ and CD8+ T cells in the presence of specific cytokines results in the induction of transcriptional programs that result in their differentiation into effector or memory cells and in the case of CD4+ T cells, the adoption of distinct T-helper fates. Previous studies have shown that histone modification and DNA methylation play important roles in each of these events. More recently, the roles of specific epigenetic regulators in T cell differentiation have been clarified. The identification of the epigenetic modifications and modifiers that control mature T cell differentiation and specification has also provided further insights into how dysregulation of these processes can lead to cancer or autoimmune diseases. In this review, we summarize recent findings that have provided new insights into epigenetic regulation of T cell differentiation in both mice and humans.

CDK6 Is a Therapeutic Target in Myelofibrosis.

Myelofibrosis (myelofibrosis) is a deadly blood neoplasia with the worst prognosis among myeloproliferative neoplasms (MPN). The JAK2 inhibitors ruxolitinib and fedratinib have been approved for treatment of myelofibrosis, but they do not offer significant improvement of bone marrow fibrosis. CDK6 expression is significantly elevated in MPN/myelofibrosis hematopoietic progenitor cells. In this study, we investigated the efficacy of CDK4/6 inhibitor palbociclib alone or in combination with ruxolitinib in Jak2V617F and MPLW515L murine models of myelofibrosis. Treatment with palbociclib alone significantly reduced leukocytosis and splenomegaly and inhibited bone marrow fibrosis in Jak2V617F and MPLW515L mouse models of myelofibrosis. Combined treatment of palbociclib and ruxolitinib resulted in normalization of peripheral blood leukocyte counts, marked reduction of spleen size, and abrogation of bone marrow fibrosis in murine models of myelofibrosis. Palbociclib treatment also preferentially inhibited Jak2V617F mutant hematopoietic progenitors in mice. Mechanistically, treatment with palbociclib or depletion of CDK6 inhibited Aurora kinase, NF-κB, and TGFß signaling pathways in Jak2V617F mutant hematopoietic cells and attenuated expression of fibrotic markers in the bone marrow. Overall, these data suggest that palbociclib in combination with ruxolitinib may have therapeutic potential for treatment of myelofibrosis and support the clinical investigation of this drug combination in patients with myelofibrosis. SIGNIFICANCE: These findings demonstrate that CDK6 inhibitor palbociclib in combination with ruxolitinib ameliorates myelofibrosis, suggesting this drug combination could be an effective therapeutic strategy against this devastating blood disorder.

U2af1 is required for survival and function of hematopoietic stem/progenitor cells.

U2AF1 is involved in the recognition of the 3' splice site during pre-mRNA splicing. Mutations in U2AF1 are frequently observed in myelodysplastic syndromes. However, the role of wild-type U2AF1 in normal hematopoiesis has remained elusive. Using a novel conditional U2af1 knockout allele, we have found that deletion of U2af1 results in profound defects in hematopoiesis characterized by pancytopenia, ablation of hematopoietic stem/progenitor cells (HSPC) leading to bone marrow failure and early lethality in mice. U2af1 deletion impairs HSPC function and repopulation capacity. U2af1 deletion also causes increased DNA damage and reduced survival in hematopoietic progenitors. RNA sequencing analysis reveals significant alterations in the expression of genes related to HSC maintenance, cell proliferation, and DNA damage response-related pathways in U2af1-deficient HSPC. U2af1 deficiency also induces splicing alterations in genes important for HSPC function. This includes altered splicing and perturbed expression of Nfya and Pbx1 transcription factors in U2af1-deficient HSPC. Collectively, these results suggest an important role for U2af1 in the maintenance and function of HSPC in normal hematopoiesis. A better understanding of the normal function of U2AF1 in hematopoiesis is important for development of appropriate therapeutic approaches for U2AF1 mutant induced hematologic malignancies.