Histone H2AX stabilizes broken DNA strands to suppress chromosome breaks and translocations during V(D)J recombination

The H2AX core histone variant is phosphorylated in chromatin around DNA double strand breaks (DSBs) and functions through unknown mechanisms to suppress antigen receptor locus translocations during V(D)J recombination. Formation of chromosomal coding joins and suppression of translocations involves the ataxia telangiectasia mutated and DNA-dependent protein kinase catalytic subunit serine/threonine kinases, each of which phosphorylates H2AX along cleaved antigen receptor loci. Using Abelson transformed pre–B cell lines, we find that H2AX is not required for coding join formation within chromosomal V(D)J recombination substrates. Yet we show that H2AX is phosphorylated along cleaved Igκ DNA strands and prevents their separation in G1 phase cells and their progression into chromosome breaks and translocations after cellular proliferation. We also show that H2AX prevents chromosome breaks emanating from unrepaired RAG endonuclease-generated TCR-α/δ locus coding ends in primary thymocytes. Our data indicate that histone H2AX suppresses translocations during V(D)J recombination by creating chromatin modifications that stabilize disrupted antigen receptor locus DNA strands to prevent their irreversible dissociation. We propose that such H2AX-dependent mechanisms could function at additional chromosomal locations to facilitate the joining of DNA ends generated by other types of DSBs

The non-peptide thrombopoietin receptor agonist eltrombopag stimulates megakaryopoiesis in bone marrow cells from patients with relapsed multiple myeloma

Background: Thrombocytopenia is a significant problem in patients with relapsed or refractory multiple myeloma, precipitating a need for supportive platelet transfusions and necessitating decreases in delivered doses of chemotherapy. Eltrombopag is a non-peptide, small molecule thrombopoietin (TPO) receptor agonist that promotes megakaryopoiesis similar to endogenous human TPO and may be an effective agent for thrombocytopenia in this patient population. Methods: We examined the effects of eltrombopag on megakaryocyte colony-forming capacity in CD34+ cells in patients with multiple myeloma and investigated its impact on proliferation, viability, and apoptosis in primary CD138+ human myeloma cells and myeloma cell lines. Results: Eltrombopag at doses of 0.1 to 100 μM did not enhance proliferation of primary human CD138+ multiple myeloma cells from patients with relapsed disease or myeloma cell lines when used alone or in combination with erythropoietin (EPO) and granulocyte colony-stimulating factor (G-CSF) and did not alter cell viability nor apoptosis of human myeloma cells exposed to bortezomib and lenalidomide. Eltrombopag stimulated megakaryopoiesis in human CD34+ cells from normal individuals and from patients with relapsed multiple myeloma via activation of Akt signaling pathways. Conclusions: These results provide proof-of-principle supporting the design of future clinical studies examining eltrombopag for the treatment of thrombocytopenia in patients with advanced multiple myeloma

Bcl-2 Overexpression Leads to Increases in Suppressor of Cytokine Signaling-3 Expression in B Cells and De Novo Follicular Lymphoma

The t(14;18)(q32;q21), resulting in deregulated expression of B-cell-leukemia/lymphoma-2 (Bcl-2), represents the genetic hallmark in human follicular lymphomas. Substantial evidence supports the hypothesis that the t(14;18) and Bcl-2 overexpression are necessary but not solely responsible for neoplastic transformation and require cooperating genetic derangements for neoplastic transformation to occur. To investigate genes that cooperate with Bcl-2 to influence cellular signaling pathways important for neoplastic transformation, we used oligonucleotide microarrays to determine differential gene expression patterns in CD19+ B cells isolated from Eμ-Bcl-2 transgenic mice and wild-type littermate control mice. Fifty-seven genes were induced and 94 genes were repressed by ≥2-fold in Eμ-Bcl-2 transgenic mice (P \u3c 0.05). The suppressor of cytokine signaling-3 (SOCS3) gene was found to be overexpressed 5-fold in B cells from Eμ-Bcl-2 transgenic mice. Overexpression of Bcl-2 in both mouse embryo fibroblast-1 and hematopoietic cell lines resulted in induction of SOCS3 protein, suggesting a Bcl-2-associated mechanism underlying SOCS3 induction. Immunohistochemistry with SOCS3 antisera on tissue from a cohort of patients with de novo follicular lymphoma revealed marked overexpression of SOCS3 protein that, within the follicular center cell region, was limited to neoplastic follicular lymphoma cells and colocalized with Bcl-2 expression in 9 of 12 de novo follicular lymphoma cases examined. In contrast, SOCS3 protein expression was not detected in the follicular center cell region of benign hyperplastic tonsil tissue. These data suggest that Bcl-2 overexpression leads to the induction of activated signal transducer and activator of transcription 3 (STAT3) and to the induction of SOCS3, which may contribute to the pathogenesis of follicular lymphoma

Pim kinases modulate resistance to tyrosine kinase inhibitors in FLT3-ITD acute myeloid leukemia

Fms-related tyrosine kinase-3 internal tandem duplication (FLT3-ITD) mutations are frequently detected in acute myeloid leukemia (AML) patients and are associated with a dismal long-term prognosis. FLT3 tyrosine kinase inhibitors (TKI) may provide efficient short-term disease control, however virtually all responding patients relapse within few months. The oncogenic Pim protein kinases are FLT3 targets expressed in AML cells but their participation in disease pathogenesis is incompletely understood. Here we show that Pim-2 overexpression is frequently found in samples from AML patients experiencing resistance to TKI therapy. Pim-2 activity is critical for tumor propagation in TKI-resistant cells and in a mouse model of FLT3-ITD-induced myeloproliferative neoplasm ectopic Pim-2 expression induces TKI resistance. Inhibition of Pim kinases enhances TKI docking on FLT3 ATPbinding pocket therefore restoring its ability to block downstream receptor signaling. Finally, combined Pim and FLT3 kinases inhibitors efficiently eradicate FLT3-mutated cell lines and primary AML samples. The implication of Pim kinases in TKI resistance open new perspectives for AML targeted therapies