Persistence of CCR5 usage among primary human immunodeficiency virus isolates of individuals receiving intermittent interleukin-2.

OBJECTIVE: To investigate the impact of intermittent interleukin-2 (IL-2) plus combination antiretroviral therapy (cART) on HIV-1 entry co-receptor use. METHODS: Primary HIV-1 isolates were obtained from 54 HIV-1-positive individuals at baseline and after 12 months using co-cultivation of peripheral blood mononuclear cells (PBMC) with activated PBMC of HIV-negative healthy donors. HIV-1 co-receptor use was determined on U87-CD4 cells. RESULTS: Fourteen out of the 21 (67%) IL-2-treated individuals harbouring a primary CCR5-dependent (R5) HIV-1 isolate at baseline confirmed an R5 virus isolation after 12 months in contrast to 3 out of 7 (43%) of those receiving cART only. After 12 months, only 1 R5X4 HIV-1 isolate was obtained from 21 cART+IL-2-treated individuals infected with an R5 virus at entry (5%) vs. 2/7 (29%) patients receiving cART alone, as confirmed by a 5-year follow-up on some individuals. CONCLUSIONS: Intermittent IL-2 administration plus cART may prevent evolution towards CXCR4 usage in individuals infected with R5 HIV-1.

Coding sequence and intron-exon junctions of the c-myb gene are intact in the chronic phase and blast crisis stages of chronic myeloid leukemia patients.

The c-myb gene encodes a transcription factor required for proliferation, differentiation and survival of normal and leukemic hematopoietic cells. c-Myb has a longer half-life in BCR/ABL-expressing than in normal cells, a feature which depends, in part, on PI-3K/Akt-dependent regulation of proteins interacting with the leucine zipper/negative regulatory region of c-Myb. Thus, we asked whether the stability of c-Myb in leukemic cells might be enhanced by mutations interfering with its degradation. We analyzed the c-myb gene in 133 chronic myeloid leukemia (CML) patients in chronic phase and/or blast crisis by denaturing-high performance liquid chromatography (D-HPLC) and sequence analysis of PCR products corresponding to the entire coding sequence and each exon-intron boundary. No mutations were found. We found four single nucleotide polymorphisms (SNPs) and identified an alternatively spliced transcript lacking exon 5, but SNPs frequency and expression of the alternatively spliced transcript were identical in normal and CML cells. Thus, the enhanced stability of c-Myb in CML blast crisis cells and perhaps in other types of leukemia is not caused by a genetic mechanism.

CDKN2A-independent role of BMI1 in promoting growth and survival of Ph+ acute lymphoblastic leukemia.

BMI1 is a key component of the PRC1 (polycomb repressive complex-1) complex required for maintenance of normal and cancer stem cells. Its aberrant expression is detected in chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia (ALL), but no data exist on BMI1 requirement in ALL cells. We show here that BMI1 expression is important for proliferation and survival of Ph+ ALL cells and for leukemogenesis of Ph+ cells in vivo. Levels of BIM, interferon-α (IFNα)-regulated genes and E2F7 were upregulated in BMI1-silenced cells, suggesting that repressing their expression is important for BMI1 biological effects. Consistent with this hypothesis, we found that: (i) downregulation of BIM or E2F7 abrogated apoptosis or rescued, in part, the reduced proliferation and colony formation of BMI1 silenced BV173 cells; (ii) BIM/E2F7 double silencing further enhanced colony formation and in vivo leukemogenesis of BMI1-silenced cells; (iii) overexpression of BIM and E2F7 mimicked the effect of BMI1 silencing in BV173 and SUP-B15 cells; and (iv) treatment with IFNα suppressed proliferation and colony formation of Ph+ ALL cells. These studies indicate that the growth-promoting effects of BMI1 in Ph+ ALL cells depend on suppression of multiple pathways and support the use of IFNα in the therapy of Ph+ ALL.

Gfi-1 inhibits proliferation and colony formation of p210BCR/ABL-expressing cells via transcriptional repression of STAT 5 and Mcl-1.

Expression of the transcription repressor Gfi-1 is required for the maintenance of murine hematopoietic stem cells. In human cells, ectopic expression of Gfi-1 inhibits and RNA interference-mediated Gfi-1 downregulation enhances proliferation and colony formation of p210BCR/ABL expressing cells. To investigate the molecular mechanisms that may explain the effects of perturbing Gfi-1 expression in human cells, Gfi-1-regulated genes were identified by microarray analysis in K562 cells expressing the tamoxifen-regulated Gfi-1-ER protein. STAT 5B and Mcl-1, two genes important for the proliferation and survival of hematopoietic stem cells, were identified as direct and functionally relevant Gfi-1 targets in p210BCR/ABL-transformed cells because: (i) their expression and promoter activity was repressed by Gfi-1 and (ii) when constitutively expressed blocked the proliferation and colony formation inhibitory effects of Gfi-1. Consistent with these findings, genetic or pharmacological inhibition of STAT 5 and/or Mcl-1 markedly suppressed proliferation and colony formation of K562 and CD34+ chronic myelogenous leukemia (CML) cells. Together, these studies suggest that the Gfi-1STAT 5B/Mcl-1 regulatory pathway identified here can be modulated to suppress the proliferation and survival of p210BCR/ABL-transformed cells including CD34+ CML cells.

Expression of p89(c-Mybex9b), an alternatively spliced form of c-Myb, is required for proliferation and survival of p210BCR/ABL-expressing cells.

The c-Myb gene encodes the p75(c-Myb) isoform and less-abundant proteins generated by alternatively spliced transcripts. Among these, the best known is p(c-Mybex9b), which contains 121 additional amino acids between exon 9 and 10, in a domain involved in protein-protein interactions and negative regulation. In hematopoietic cells, expression of p(c-Mybex9b) accounts for 10-15% of total c-Myb; these levels may be biologically relevant because modest changes in c-Myb expression affects proliferation and survival of leukemic cells and lineage choice and frequency of normal hematopoietic progenitors. In this study, we assessed biochemical activities of p(c-Mybex9b) and the consequences of perturbing its expression in K562 and primary chronic myeloid leukemia (CML) progenitor cells. Compared with p75(c-Myb), p(c-Mybex9b) is more stable and more effective in transactivating Myb-regulated promoters. Ectopic expression of p(c-Mybex9b) enhanced proliferation and colony formation and reduced imatinib (IM) sensitivity of K562 cells; conversely, specific downregulation of p(c-Mybex9b) reduced proliferation and colony formation, enhanced IM sensitivity of K562 cells and markedly suppressed colony formation of CML CD34(+) cells, without affecting the levels of p75(c-Myb). Together, these studies indicate that expression of the low-abundance p(c-Mybex9b) isoform has an important role for the overall biological effects of c-Myb in BCR/ABL-transformed cells.