Class II Transactivator (CIITA) Enhances Cytoplasmic Processing of HIV-1 Pr55Gag

The Pr55(gag) (Gag) polyprotein of HIV serves as a scaffold for virion assembly and is thus essential for progeny virion budding and maturation. Gag localizes to the plasma membrane (PM) and membranes of late endosomes, allowing for release of infectious virus directly from the cell membrane and/or upon exocytosis. The host factors involved in Gag trafficking to these sites are largely unknown. Upon activation, CD4+ T cells, the primary target of HIV infection, express the class II transcriptional activator (CIITA) and therefore the MHC class II isotype, HLA-DR. Similar to Gag, HLA-DR localizes to the PM and at the membranes of endosomes and specialized vesicular MHC class II compartments (MIICs). In HIV producer cells, transient HLA-DR expression induces intracellular Gag accumulation and impairs virus release.Here we demonstrate that both stable and transient expression of CIITA in HIV producer cells does not induce HLA-DR-associated intracellular retention of Gag, but does increase the infectivity of virions. However, neither of these phenomena is due to recapitulation of the class II antigen presentation pathway or CIITA-mediated transcriptional activation of virus genes. Interestingly, we demonstrate that CIITA, apart from its transcriptional effects, acts cytoplasmically to enhance Pr160(gag-pol) (Gag-Pol) levels and thereby the viral protease and Gag processing, accounting for the increased infectivity of virions from CIITA-expressing cells.This study demonstrates that CIITA enhances HIV Gag processing, and provides the first evidence of a novel, post-transcriptional, cytoplasmic function for a well-known transactivator

High CIP2A levels correlate with an antiapoptotic phenotype that can be overcome by targeting BCL-XL in chronic myeloid leukemia. Leukemia

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a predictive biomarker of disease progression in many malignancies, including imatinib-treated chronic myeloid leukemia (CML). Although high CIP2A levels correlate with disease progression in CML, the underlying molecular mechanisms remain elusive. In a screen of diagnostic chronic phase samples from patients with high and low CIP2A protein levels, high CIP2A levels correlate with an antiapoptotic phenotype, characterized by downregulation of proapoptotic BCL-2 family members, including BIM, PUMA and HRK, and upregulation of the antiapoptotic protein BCL-XL. These results suggest that the poor prognosis of patients with high CIP2A levels is due to an antiapoptotic phenotype. Disrupting this antiapoptotic phenotype by inhibition of BCL-XL via RNA interference or A-1331852, a novel, potent and BCL-XL-selective inhibitor, resulted in extensive apoptosis either alone or in combination with imatinib, dasatinib or nilotinib, both in cell lines and in primary CD34(+) cells from patients with high levels of CIP2A. These results demonstrate that BCL-XL is the major antiapoptotic survival protein and may be a novel therapeutic target in CML

STAT3 Contributes to Resistance Towards BCR-ABL Inhibitors in a Bone Marrow Microenvironment Model of Drug Resistance in Chronic Myeloid Leukemia Cells

Imatinib mesylate (imatinib) represents a potent molecularly targeted therapy against the oncogenic tyrosine kinase, BCR-ABL. Although imatinib has shown considerable efficacy against chronic myeloid leukemia (CML), displaying high rates of complete hematological and complete cytogenetic responses, treatment with imatinib is not curative and overtime advanced-stage CML patients often become refractory to further treatment. Acquired resistance to imatinib has been associated with mutations within the kinase domain of BCR-ABL, BCR-ABL gene amplification, leukemic stem cell quiescence as well as over-expression of the multidrug resistance (MDR1) gene. However, in vitro resistance models often fail to consider the role of the tumor microenvironment in the emergence of the imatinib-resistant phenotype. The bone marrow is the predominant microenvironment of CML and is a rich source of both soluble factors and extracellular matrixes, which may influence drug response. To address the influence of the bone marrow microenvironment on imatinib sensitivity, we utilized an in vitro co-culture bone marrow stroma model. Using a transwell system, we demonstrated that soluble factors secreted by the human bone marrow stroma cell line, HS-5, were sufficient to cause resistance to apoptosis induced by imatinib in CML cell lines. We subsequently determined that culturing CML cells in HS-5-derived conditioned media (CM) inhibits apoptosis induced by imatinib and other second generation BCR-ABL inhibitors. These data suggest that more potent BCR-ABL inhibitors will not overcome resistance associated with the bone marrow microenvironment. Additionally, we determined that CM increases the clonogenic survival of CML cells following treatment with imatinib. HS-5 cells are reported to express several cytokines and growth factors known to activate signal transducer and activator of transcription 3 (STAT3). Given its crucial role in the survival of hematopoietic cells, we asked whether, 1) CM derived from HS-5 cells can activate STAT3 in CML cells and 2) does activation of STAT3 confer resistance to BCR-ABL inhibitors. We demonstrated that exposure of the CML cell lines, K562 and KU812, to CM caused an increase in phospho-Tyr STAT3, while no increases in phospho-Tyr STAT5 were noted. Moreover, resistance was associated with increased levels of the STAT3 target genes, Bcl-xl, Mcl-1 and survivin. Furthermore, reducing STAT3 levels with siRNA sensitized K562 cells cultured in CM to imatinib-induced cell death (p\u3c0.05, Student’s t-test). Importantly, STAT3 dependency was specific for cells grown in CM, as reducing STAT3 levels in regular growth conditions had no effect on imatinib sensitivity. Together, these data support a novel mechanism of BCR-ABL-independent imatinib resistance and provide preclinical rationale for using STAT3 inhibitors to increase the efficacy of imatinib within the context of the bone marrow microenvironment

STAT3 Contributes to Resistance Towards BCR-ABL Inhibitors in a Bone Marrow Microenvironment Model of Drug Resistance in Chronic Myeloid Leukemia Cells

Imatinib mesylate (imatinib) represents a potent molecularly targeted therapy against the oncogenic tyrosine kinase, BCR-ABL. Although imatinib has shown considerable efficacy against chronic myeloid leukemia (CML), displaying high rates of complete hematological and complete cytogenetic responses, treatment with imatinib is not curative and overtime advanced-stage CML patients often become refractory to further treatment. Acquired resistance to imatinib has been associated with mutations within the kinase domain of BCR-ABL, BCR-ABL gene amplification, leukemic stem cell quiescence as well as over-expression of the multidrug resistance (MDR1) gene. However, in vitro resistance models often fail to consider the role of the tumor microenvironment in the emergence of the imatinib-resistant phenotype. The bone marrow is the predominant microenvironment of CML and is a rich source of both soluble factors and extracellular matrixes, which may influence drug response. To address the influence of the bone marrow microenvironment on imatinib sensitivity, we utilized an in vitro co-culture bone marrow stroma model. Using a transwell system, we demonstrated that soluble factors secreted by the human bone marrow stroma cell line, HS-5, were sufficient to cause resistance to apoptosis induced by imatinib in CML cell lines. We subsequently determined that culturing CML cells in HS-5-derived conditioned media (CM) inhibits apoptosis induced by imatinib and other second generation BCR-ABL inhibitors. These data suggest that more potent BCR-ABL inhibitors will not overcome resistance associated with the bone marrow microenvironment. Additionally, we determined that CM increases the clonogenic survival of CML cells following treatment with imatinib. HS-5 cells are reported to express several cytokines and growth factors known to activate signal transducer and activator of transcription 3 (STAT3). Given its crucial role in the survival of hematopoietic cells, we asked whether, 1) CM derived from HS-5 cells can activate STAT3 in CML cells and 2) does activation of STAT3 confer resistance to BCR-ABL inhibitors. We demonstrated that exposure of the CML cell lines, K562 and KU812, to CM caused an increase in phospho-Tyr STAT3, while no increases in phospho-Tyr STAT5 were noted. Moreover, resistance was associated with increased levels of the STAT3 target genes, Bcl-xl, Mcl-1 and survivin. Furthermore, reducing STAT3 levels with siRNA sensitized K562 cells cultured in CM to imatinib-induced cell death (p\u3c0.05, Student’s t-test). Importantly, STAT3 dependency was specific for cells grown in CM, as reducing STAT3 levels in regular growth conditions had no effect on imatinib sensitivity. Together, these data support a novel mechanism of BCR-ABL-independent imatinib resistance and provide preclinical rationale for using STAT3 inhibitors to increase the efficacy of imatinib within the context of the bone marrow microenvironment