José A. García-Marco

α4β1 integrin and 190-kDa CD44v constitute a cell surface docking complex for gelatinase B/MMP-9 in chronic leukemic but not in normal B cells

As B-cell chronic lymphocytic leukemia (B-CLL) progresses, malignant cells extravasate and infiltrate lymphoid tissues. Several molecules, including gelatinase B/MMP-9, contribute to these processes. Although mainly a secreted protease, some MMP-9 is present at the B-CLL cell surface and the function, mode of anchoring, and interactions of this MMP-9 are unknown. Here we show that anti-MMP-9 antibodies immunoprecipitated a 190-kDa CD44v isoform and alpha4beta1 integrin from B-CLL cells, but not from normal B cells. Function-blocking antibodies to alpha4beta1 or CD44, or transfection with specific siRNAs, decreased cell-associated proMMP-9 and increased the secreted form. B-CLL cells attached to and bound proMMP-9 and active MMP-9, and this was inhibited by blocking the expression or function of alpha4beta1 or CD44. The MMP-9 hemopexin domain was critical in these interactions. alpha4beta1 and 190-kDa CD44v (but not CD44H) formed a complex at the cell surface, since they both coimmunoprecipitated with anti-alpha4, anti-beta1, or anti-CD44 antibodies. Immunofluorescence analyses confirmed that alpha4beta1 and CD44v colocalized with MMP-9. Binding of proMMP-9 inhibited B-CLL cell migration, and this required MMP-9 proteolytic activity. Thus, we have identified alpha4beta1 and CD44v as a novel proMMP-9 cell surface docking complex and show that cell-associated MMP-9 may regulate B-CLL cell migration and arrest.

Matrix Metalloproteinase-9 Promotes Chronic Lymphocytic Leukemia B Cell Survival through Its Hemopexin Domain

Matrix metalloproteinase-9 (MMP-9) is the major MMP produced by B-CLL cells and contributes to their tissue infiltration by degrading extracellular and membrane-anchored substrates. Here we describe a different function for MMP-9 in B-CLL, which involves the hemopexin domain rather than its catalytic function. Binding of soluble or immobilized (pro)MMP-9, a catalytically inactive proMMP-9 mutant, or the MMP-9 hemopexin domain to its docking receptors alpha4beta1 integrin and CD44v, induces an intracellular signaling pathway that prevents B-CLL apoptosis. This pathway is induced in all B-CLL cases, is active in B-CLL lymphoid tissues, and consists of Lyn activation, STAT3 phosphorylation, and Mcl-1 upregulation. Our results establish that MMP/receptor binding induces intracellular survival signals and highlight the role of (pro)MMP-9 in B-CLL pathogenesis.

PLCG1 mutations in cutaneous T-cell lymphomas

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of primary cutaneous T-cell lymphoproliferative processes, mainly composed of mycosis fungoides and Sézary syndrome, the aggressive forms of which lack an effective treatment. The molecular pathogenesis of CTCL is largely unknown, although neoplastic cells show increased signaling from T-cell receptors (TCRs). DNAs from 11 patients with CTCL, both normal and tumoral, were target-enriched and sequenced by massive parallel sequencing for a selection of 524 TCR-signaling-related genes. Identified variants were validated by capillary sequencing. Multiple mutations were found that affected several signaling pathways, such as TCRs, nuclear factor κB, or Janus kinase/signal transducer and activator of transcription, but PLCG1 was found to be mutated in 3 samples, 2 of which featured a redundant mutation (c.1034T>C, S345F) in exon 11 that affects the PLCx protein catalytic domain. This mutation was further analyzed by quantitative polymerase chain reaction genotyping in a new cohort of 42 patients with CTCL, where it was found in 19% of samples. Immunohistochemical analysis for nuclear factor of activated T cells (NFAT) showed that PLCG1-mutated cases exhibited strong NFAT nuclear immunostaining. Functional studies demonstrated that PLCG1 mutants elicited increased downstream signaling toward NFAT activation, and inhibition of this pathway resulted in reduced CTCL cell proliferation and cell viability. Thus, increased proliferative and survival mechanisms in CTCL may partially depend on the acquisition of somatic mutations in PLCG1 and other genes that are essential for normal T-cell differentiation.

Engagement of α4β1 integrin by fibronectin induces in vitro resistance of B chronic lymphocytic leukemia cells to fludarabine

B‐cell chronic lymphocytic leukemia is characterized by the accumulation of malignant B lymphocytes as a result of abnormal survival signals operating in vivo. Previously, we showed that adhesion of B‐CLL cells to the fibronectin fragment H89, a ligand for α4β1 integrin, prevents their spontaneous apoptosis in vitro. We have now studied whether α4β1/H89 interaction affected the response of B‐CLL cells to the therapeutic drug fludarabine. B‐CLL cells cultured on H89 during treatment with fludarabine showed significantly higher mean viability (P<0.05) than cells cultured on the control polylysine for all doses of drug tested. Similar results were obtained with the EHEB cell line. Analysis of the expression of Bcl‐2‐family proteins after 48 h of fludarabine treatment revealed that Bcl‐xL levels were significantly higher (P<0.05) for cells cultured on H89 than on polylysine and correlated (r=0.56,P<0.05) with the increased cell viability observed on H89 cultures. These results indicate that Bcl‐xL is involved in the survival signals induced by α4β1 ligation and may contribute to the progressive drug resistance observed in B‐CLL.

TCL1A expression delineates biological and clinical variability in B-cell lymphoma.

The assembly of a collection of gene-expression signatures of the major types of B-cell non-Hodgkins lymphoma has identified increased T-cell leukemia/lymphoma 1A (TCL1) expression in multiple lymphoma types and cases, and has enabled the investigation of the functional and clinical importance of TCL1 expression. Specifically, Burkitts lymphoma cases show a homogeneously strong expression of TCL1, whereas diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, nodal marginal zone lymphoma, and splenic marginal zone lymphoma display a striking variability in the intensity of TCL1 staining. This was validated in two independent series. A Gene-Set Enrichment Analysis of the genes correlated with TCL1A expression found that variation in the level of expression of TCL1A was significantly associated with some of the most important gene signatures recognizing B-cell lymphoma pathogenesis and heterogeneity, such as germinal center, B-cell receptor, NF-κB (and its target genes), death, MAP kinases, TNFR1, TOLL, and IL1R. Additionally, TCL1 expression was correlated with shorter time to treatment in chronic lymphocytic leukemia cases and shorter lymphoma-specific survival in mantle cell lymphoma series, thus indicating the clinical and biological significance of TCL1 expression, and suggesting TCL1A as a potential therapeutic target.

The class II tumor-suppressor gene RARRES3 is expressed in B cell lymphocytic leukemias and down-regulated with disease progression

The molecular pathogenesis of B cell chronic lymphocytic leukemia (B-CLL), the most common form of leukemia, remains unknown. We have used the mRNA differential display technique to analyze genes that may be involved in the development/progression of B-CLL. We have identified the tumor suppressor retinoic acid receptor responder 3 (RARRES3) as a B-CLL-related gene. RARRES3 maps to chromosome band 11q23, a region frequently deleted in lymphoproliferative disorders. To assess the potential involvement of RARRES3 in leukemogenesis, we examined 24 cases of B-CLL, 10 of acute lymphocytic leukemia (ALL) and five related cell lines by RT-PCR and sequence analyses. We report a correlation between RARRES3 down-regulation and B-CLL progression. We also found decreased RARRES3 gene levels in ALL cases and in the five cell lines studied. We did not find mutations in any of the leukemia samples assayed, including those with 11q23 deletion. These results indicate that RARRES3 may play a role in B-CLL progression.

AT514, a cyclic depsipeptide from Serratia marcescens, induces apoptosis of B-chronic lymphocytic leukemia cells: interference with the Akt/NF-κB survival pathway

Clinical treatment of B-cell chronic lymphocytic leukemia (B-CLL) is limited by the progressive drug resistance and nonselectivity of most drugs towards malignant cells. Depsipeptides are present in certain bacteria and display potent antitumor activity. We have studied the effect of the novel cyclodepsipeptide AT514 (serratamolide) from Serratia marcescens on B-CLL cell viability. AT514 induced apoptosis of B-CLL cells from the 21 patients studied, as confirmed by Annexin-V binding and nuclei condensation, with an average IC50 of 13 μM. AT514 was effective in those B-CLL cases resistant to fludarabine, but had no effect on normal PBL. AT514 preferentially activated the intrinsic apoptotic pathway, as evidenced by loss of mitochondrial membrane potential, release of cytochrome c and activation of caspase-9 and -3, but not of caspase-8. Importantly, AT514 interfered with phosphatidylinositol-3 kinase and protein kinase C survival signals since it increased the apoptotic effect of LY294002 and BisI inhibitors, and induced Akt dephosphorylation at Ser 473. AT514 also decreased NF-κB activity by dramatically reducing the levels of p65 in B-CLL. This was confirmed on functional assays using NF-κB-luc-transfected Raji cells and transgenic mice. Our results establish that AT514 induces apoptosis of primary B-CLL cells and could be useful for clinical treatment of this malignancy.

Efficacy and safety of liposomal cytarabine in lymphoma patients with central nervous system involvement from lymphoma.

Standard intrathecal chemotherapy for lymphomatous meningitis (LM) is limited by the short cerebrospinal half‐lives of the agents used, necessitating frequent administration. Liposomal cytarabine (DepoCyte) has an extended half‐life that permits administration at 2‐ to 4‐weekly intervals.

Long-term follow-up of dose-adjusted EPOCH plus rituximab (DA-EPOCH-R) in untreated patients with poor prognosis large B-cell lymphoma. A phase II study conducted by the Spanish PETHEMA Group

This prospective multi‐institutional phase II study was designed to assess the efficacy and safety of dose‐adjusted EPOCH (etoposide, prednisone, vincristine, cyclophosphamide and doxorubicin) plus rituximab (DA‐EPOCH‐R) in untreated patients with poor prognosis large B‐cell lymphomas. Eighty‐one patients diagnosed with diffuse large B‐cell lymphoma (DLBCL, n = 68), primary mediastinal DLBCL (n = 6) and follicular lymphoma Grade 3b (n = 7), with an age‐adjusted International Prognostic Index >1, were eligible for analysis. Median age was 60 years (range: 21–77). Sixty‐five patients (80·2%) achieved complete response. After a median follow‐up time of 64 months, 10‐year event‐free survival and overall survival (OS) were 47·8% and 63·6%, respectively. None of the studied clinical and biological characteristics were associated with poorer outcome. Interestingly, patients with BCL6 rearrangement achieved a 10‐year OS of 100%, while patients with BCL2 rearrangement exhibited a poorer outcome compared to activated B‐cell tumours and germinal centre B‐cell without BCL2 rearranged tumours. Results achieved with DA‐EPOCH‐R showed a good long‐term outcome and a tolerable toxicity profile in high‐risk large B cell lymphoma patients. Outcome was not affected by tumour cell proliferation or by cell of origin, highlighting the requirement of new biological markers for patient subclassification of high‐risk DLBCL patients.

Randomized phase 2 study of otlertuzumab and bendamustine versus bendamustine in patients with relapsed chronic lymphocytic leukaemia

Otlertuzumab (TRU‐016) is a humanized anti‐CD37 protein therapeutic that triggers direct caspase‐independent apoptosis of malignant B cells and induces antibody‐dependent cell‐mediated cytotoxicity. Patients with relapsed chronic lymphocytic leukaemia (CLL) received either otlertuzumab (20 mg/kg) weekly by IV infusion for two 28‐day cycles then every 14 days for four 28‐day cycles and IV bendamustine (70 mg/m2) on Days 1 and 2 of each cycle for up to six 28‐day cycles or bendamustine alone. Thirty‐two patients were treated with otlertuzumab and bendamustine and 33 with bendamustine alone. Overall response rate according to the International Workshop on Chronic Lymphocytic Leukaemia criteria was 69% in the otlertuzumab and bendamustine arm and 39% in the bendamustine alone arm (P = 0·025). Median progression‐free survival (PFS) was 15·9 months in the otlertuzumab and bendamustine arm and 10·2 months in the bendamustine alone arm (P = 0·0192). There was a higher incidence of pyrexia (34% vs. 12%) and neutropenia (59% vs. 39%) with the combination but this did not result in a higher incidence of severe (grade 3/4) infections (13% vs. 27%). This combination significantly increased the response rate and prolonged the PFS over single agent bendamustine in patients with relapsed or refractory CLL.