SPARC expression in CML is associated to imatinib treatment and to inhibition of leukemia cell proliferation

BACKGROUND: SPARC is a matricellular glycoprotein with growth-inhibitory and antiangiogenic activity in some cell types. The study of this protein in hematopoietic malignancies led to conflicting reports about its role as a tumor suppressor or promoter, depending on its different functions in the tumor microenvironment. In this study we investigated the variations in SPARC production by peripheral blood cells from chronic myeloid leukemia (CML) patients at diagnosis and after treatment and we identified the subpopulation of cells that are the prevalent source of SPARC. METHODS: We evaluated SPARC expression using real-time PCR and western blotting. SPARC serum levels were detected by ELISA assay. Finally we analyzed the interaction between exogenous SPARC and imatinib (IM), in vitro, using ATP-lite and cell cycle analysis. RESULTS: Our study shows that the CML cells of patients at diagnosis have a low mRNA and protein expression of SPARC. Low serum levels of this protein are also recorded in CML patients at diagnosis. However, after IM treatment we observed an increase of SPARC mRNA, protein, and serum level in the peripheral blood of these patients that had already started at 3 months and was maintained for at least the 18 months of observation. This SPARC increase was predominantly due to monocyte production. In addition, exogenous SPARC protein reduced the growth of K562 cell line and synergized in vitro with IM by inhibiting cell cycle progression from G1 to S phase. CONCLUSION: Our results suggest that low endogenous SPARC expression is a constant feature of BCR/ABL positive cells and that IM treatment induces SPARC overproduction by normal cells. This exogenous SPARC may inhibit CML cell proliferation and may synergize with IM activity against CML

Bortezomib modulates CHIT1 and YKL40 in monocyte-derived osteoclast and in myeloma cells

Osteolytic bone disease is a common manifestation of multiple myeloma (MM) that leads to progressive skeleton destruction and is the most severe cause of morbidity in MM patients.It results from increased osteolytic activity and decrease osteoblastic function. Activation of mammalian chitinases CHIT1 and YKL40 is associated with osteoclast (OCs) differentiation and bone digestion. In the current study, we investigated the effect of two Bortezomib’s concentration (BO) (2.5 nM and 5nM) on osteoclastogenesis by analyzing regulation of chitinase expression. OCs exposition to BO was able to inhibit the expression of different OCs markers such as RANK, CTSK, TRAP and MMP9. In addition BO-treatment reduced CHIT1 enzymatic activity and both CHIT1 and YKL40 mRNA expression levels and cytoplasmatic and secreted protein. Moreover, immunofluorescence evaluation of mature OCs showed that BO was able to translocate YKL40 into the nucleus, while CHIT1 remained into the cytoplasm. Since MM cell lines such as U266, SKM-M1 and MM1 showed high levels of CHIT1 activity, we analyzed bone resorption ability of U266 using dentin disc assay resorption pits. Silencing chitinase proteins in U266 cell line with specific siRNAs, resulted in pits number reduction on dentine discs. In conclusion, we showed that BO decreases osteoclastogenesis and reduces bone resorption in OCs and U266 cell line by modulating the chitinases CHIT1 and YKL40. These results indicate that chitinases may be a therapeutic target for bone disease in MM patients

The genotype of MLH1 identifies a subgroup of follicular lymphoma patients who do not benefit from doxorubicin: FIL-FOLL study

Though most follicular lymphoma biomarkers rely on tumor features, the host genetic background may also be relevant for outcome. Here we aimed at verifying the contribution of candidate polymorphisms of FCγ receptor, DNA repair and detoxification genes to prognostic stratification of follicular lymphoma treated with immunochemotherapy. The study was based on 428 patients enrolled in the FOLL05 prospective trial that compared three standard-of-care regimens (rituximab-cyclophosphamide-vincristine-prednisone versus rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone versus rituximab-fludarabine-mitoxantrone) for the first line therapy of advanced follicular lymphoma. Polymorphisms were genotyped on peripheral blood DNA samples. The primary endpoint was time to treatment failure. Polymorphisms of FCGR2A and FCGR3A, which have been suggested to influence the activity of rituximab as a single agent, did not affect time to treatment failure in the pooled analysis of the three FOLL05 treatment arms that combined rituximab with chemotherapy (P=0.742, P=0.252, respectively). These results were consistent even when the analysis was conducted by intention to treat, indicating that different chemotherapy regimens and loads did not interact differentially with the FCGR2A and FCGR3A genotypes. The genotype of MLH1, which regulates the genotoxic effect of doxorubicin, significantly affected time to treatment failure in patients in the rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone arm (P=0.001; q<0.1), but not in arms in which patients did not receive doxorubicin (i.e., the rituximab-cyclophosphamide-vincristine-prednisone and rituximab-fludarabine-mitoxantrone arms). The impact of MLH1 on time to treatment failure was independent after adjusting for the Follicular Lymphoma International Prognostic Index and other potential confounding variables by multivariate analysis. These data indicate that MLH1 genotype is a predictor of failure to benefit from rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone treatment in advanced follicular lymphoma and confirm that FCGR2A and FCGR3A polymorphisms have no impact when follicular lymphoma is treated with rituximab plus chemotherapy (clinicaltrials.gov identifier: NCT00774826)

Chk1 Inhibition Restores Inotuzumab Ozogamicin Citotoxicity in CD22-Positive Cells Expressing Mutant p53

Inotuzumab ozogamicin (IO) is an anti-CD22 calicheamicin immunoconjugate that has been recently approved for the treatment of relapsed or refractory B-Acute Lymphoblastic Leukemia (r/r B-ALL). We employed both immortalized and primary cells derived from CD22-positive lymphoproliferative disorders to investigate the signaling pathways contributing to IO sensitivity or resistance. We found that the drug reduced the proliferation rate of CD22-positive cell lines expressing wild-type p53, but was remarkably less effective on cells exhibiting mutant p53. In addition, CD22-positive cells surviving IO were mostly blocked in the G2/M phase of the cell cycle because of Chk1 activation that, in the presence of a wild-type p53 background, led to p21 induction. When we combined IO with the Chk1 inhibitor UCN-01, we successfully abrogated IO-induced G2/M arrest regardless of the underlying p53 status, indicating that the DNA damage response triggered by IO is also modulated by p53-independent mechanisms. To establish a predictive value for p53 in determining IO responsiveness, we expressed mutant p53 in cell lines displaying the wild-type gene and observed an increase in IO IC50 values. Likewise, overexpression of an inducible wild-type p53 in cells natively presenting a mutant protein decreased their IC50 for IO. These results were also confirmed in primary CD22-positive cells derived from B-ALL patients at diagnosis and from patients with r/r B-ALL. Furthermore, co-treatment with IO and UCN-01 significantly increased cell death in primary cells expressing mutant p53. In summary, our findings suggest that p53 status may represent a biomarker predictive of IO efficacy in patients diagnosed with CD22-positive malignancies

SPARC expression in CML is associated to imatinib treatment and to inhibition of leukemia cell proliferation

Abstract Background SPARC is a matricellular glycoprotein with growth-inhibitory and antiangiogenic activity in some cell types. The study of this protein in hematopoietic malignancies led to conflicting reports about its role as a tumor suppressor or promoter, depending on its different functions in the tumor microenvironment. In this study we investigated the variations in SPARC production by peripheral blood cells from chronic myeloid leukemia (CML) patients at diagnosis and after treatment and we identified the subpopulation of cells that are the prevalent source of SPARC. Methods We evaluated SPARC expression using real-time PCR and western blotting. SPARC serum levels were detected by ELISA assay. Finally we analyzed the interaction between exogenous SPARC and imatinib (IM), in vitro, using ATP-lite and cell cycle analysis. Results Our study shows that the CML cells of patients at diagnosis have a low mRNA and protein expression of SPARC. Low serum levels of this protein are also recorded in CML patients at diagnosis. However, after IM treatment we observed an increase of SPARC mRNA, protein, and serum level in the peripheral blood of these patients that had already started at 3 months and was maintained for at least the 18 months of observation. This SPARC increase was predominantly due to monocyte production. In addition, exogenous SPARC protein reduced the growth of K562 cell line and synergized in vitro with IM by inhibiting cell cycle progression from G1 to S phase. Conclusion Our results suggest that low endogenous SPARC expression is a constant feature of BCR/ABL positive cells and that IM treatment induces SPARC overproduction by normal cells. This exogenous SPARC may inhibit CML cell proliferation and may synergize with IM activity against CML.</p

Myeloid derived suppressor cells (MDSCs) are increased and exert immunosuppressive activity together with polymorphonuclear leukocytes (PMNs) in chronic myeloid leukemia patients.

Tumor immune tolerance can derive from the recruitment of suppressor cell population, including myeloid derived suppressor cells (MDSCs), able to inhibit T cells activity. We identified a significantly expanded MDSCs population in chronic myeloid leukemia (CML) patients at diagnosis that decreased to normal levels after imatinib therapy. In addition, expression of arginase 1 (Arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function, resulted in an increase in patients at diagnosis. Purified CML CD11b+CD33+CD14-HLADR- cells markedly suppressed normal donor T cell proliferation in vitro. Comparing CML Gr-MDSCs to autologous polymorphonuclear leukocytes (PMNs) we observed a higher Arg1 expression and activity in PMNs, together with an inhibitory effect on T cells in vitro. Our data indicate that CML cells create an immuno-tolerant environment associated to MDSCs expansion with immunosuppressive capacity mediated by Arg1. In addition, we demonstrated for the first time also an immunosuppressive activity of CML PMNs, suggesting a strong potential immune escape mechanism created by CML cells, which control the anti-tumor reactive T cells. MDSCs should be monitored in imatinib discontinuation trials to understand their importance in relapsing patients