The NEDD8-activating enzyme inhibitor MLN4924 induces DNA damage in Ph+ leukemia and sensitizes for ABL kinase inhibitors.

The BCR-ABL1 fusion gene is the driver oncogene in chronic myeloid leukemia (CML) and Philadelphia-chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). The introduction of tyrosine kinase inhibitors (TKIs) targeting the ABL kinase (such as imatinib) has dramatically improved survival of CML and Ph+ ALL patients. However, primary and acquired resistance to TKIs remains a clinical challenge. Ph+ leukemia patients who achieve a complete cytogenetic (CCR) or deep molecular response (MR) (≥4.5log reduction in BCR-ABL1 transcripts) represent long-term survivors. Thus, the fast and early eradication of leukemic cells predicts MR and is the prime clinical goal for these patients. We show here that the first-in-class inhibitor of the Nedd8-activating enzyme (NAE1) MLN4924 effectively induced caspase-dependent apoptosis in Ph+ leukemia cells, and sensitized leukemic cells for ABL tyrosine kinase inhibitors (TKI) and hydroxyurea (HU). We demonstrate that MLN4924 induced DNA damage in Ph+ leukemia cells by provoking the activation of an intra S-phase checkpoint, which was enhanced by imatinib co-treatment. The combination of MLN4924 and imatinib furthermore triggered a dramatic shift in the expression of MCL1 and NOXA. Our data offers a clear rationale to explore the clinical activity of MLN4924 (alone and in combination with ABL TKI) in Ph+ leukemia patients.

Dasatinib in combination with fludarabine in patients with refractory chronic lymphocytic leukemia: a multicenter phase 2 study.

Resistance to chemotherapy-induced apoptosis in CLL is associated with overexpression of antiapoptotic proteins induced by signals from the microenvironment. In vitro, dasatinib effectively inhibits expression of anti-apoptotic regulators and restores fludarabine sensitivity in activated CLL. The aim of this study was to evaluate efficacy of one cycle of dasatinib monotherapy (100mg/day, days 1-28) followed by combination of dasatinib with fludarabine (40mg/m²/day, days 1-3 every 28 day) for a total of 6 cycles in fludarabine-refractory CLL. The primary endpoint was overall response rate according to the IWCLL'08 criteria. 20 patients were enrolled: 18 completed at least one cycle of treatment of which 67% finished at least 2 cycles of combination treatment. 3 of these 18 patients reached a formal PR (16.7%). Majority of patients obtained some reduction in lymph node (LN) size. Most frequent toxicity was related to myelosuppression. NF-κB RNA expression levels of circulating CLL cells decreased whereas the levels of pro-apoptotic NOXA increased during treatment. In conclusion, dasatinib/fludarabine combination has modest clinical efficacy in fludarabine-refractory patients.

Chronic lymphocytic leukemia disease progression is accelerated by APRIL-TACI interaction in the TCL1 transgenic mouse model.

Although in vitro studies pointed to the tumor necrosis factor family member APRIL (a proliferation-inducing ligand) in mediating survival of chronic lymphocytic leukemia (CLL) cells, clear evidence for a role in leukemogenesis and progression in CLL is lacking. APRIL significantly prolonged in vitro survival of CD5(+)B220(dull) leukemic cells derived from the murine Eµ-TCL1-Tg (TCL1-Tg [transgenic]) model for CLL. APRIL-TCL1 double-Tg mice showed a significantly earlier onset of leukemia and disruption of splenic architecture, and survival was significantly reduced. Interestingly, clonal evolution of CD5(+)B220(dull) cells (judged by BCR clonality) did not seem to be accelerated by APRIL; both mouse strains were oligoclonal at 4 months. Although APRIL binds different receptors, APRIL-mediated leukemic cell survival depended on tumor necrosis factor receptor superfamily member 13B (TACI) ligation. These findings indicate that APRIL has an important role in CLL and that the APRIL-TACI interaction might be a selective novel therapeutic target for human CLL.

IL-21 and CD40L signals from autologous T cells can induce antigen-independent proliferation of CLL cells.

Chronic lymphocytic leukemia (CLL) cells multiply in secondary lymphoid tissue, but the mechanisms leading to their proliferation are still uncertain. In addition to B-cell receptor (BCR)-triggered signals, other microenvironmental factors might well be involved. In proliferation centers, leukemic B cells are in close contact with CD4(+)CD40L(+) T cells. Therefore, we here dissected the signals provided by autologous activated T cells (Tact) to CLL cells. Although the gene expression profile induced by Tact was highly similar to that induced by sole CD40 signaling, an obvious difference was that Tact induced proliferation of CLL cells. We determined that stimulation with only CD40L+IL-21 was sufficient to induce robust proliferation in CLL cells. We then defined an interleukin (IL)-21-induced gene signature in CLL, containing components of Janus kinase/signal transducer and activator of transcription and apoptosis pathways, and this signature could be detected in lymph node (LN) samples from patients. Finally, we could detect IL-21 RNA and protein in LN, and IL-21 production ex vivo by LN CD4(+)CXCR5(+) follicular helper T cells. These results indicate that in addition to BCR signaling, activated T cells might contribute to CLL cell proliferation via CD40 and IL-21. Targeting these signaling pathways might offer new venues for treatment of CLL.

Tipping the Noxa/Mcl-1 balance overcomes ABT-737 resistance in chronic lymphocytic leukemia.

PURPOSE: Chronic lymphocytic leukemia (CLL) cells in lymph nodes (LN), from which relapses are postulated to originate, display an antiapoptotic profile in contrast to CLL cells from peripheral blood (PB). The BH3 mimetic ABT-737 antagonizes the antiapoptotic proteins Bcl-X(L) and Bcl-2 but not Mcl-1 or Bfl-1. Previously, it was shown that CD40-stimulated CLL cells were resistant to ABT-737. We aimed to define which antiapoptotic proteins determine resistance to ABT-737 in CLL and whether combination of known antileukemia drugs and ABT-737 was able to induce apoptosis of CD40-stimulated CLL cells. EXPERIMENTAL DESIGN: To mimic the LN microenvironment, PB lymphocytes of CLL patients were cultured on feeder cells expressing CD40L and treated with ABT-737 with or without various drugs. In addition, we carried out overexpression or knockdown of pro- and antiapoptotic proteins in immortalized primary B cells. RESULTS: Upon CD40 stimulation patient-specific variations in ABT-737 sensitivity correlated with differences in levels of Mcl-1 and its antagonist Noxa. Knockdown of Noxa, as well as Mcl-1 overexpression, corroborated the importance of the Noxa/Mcl-1 ratio in determining the response to ABT-737. Inhibition of NF-κB resulted in increased Noxa levels and enhanced sensitivity to ABT-737. Interestingly, increasing the Noxa/Mcl-1 ratio, by decreasing Mcl-1 (dasatinib and roscovitine) or increasing Noxa levels (fludarabine and bortezomib), resulted in synergy with ABT-737. CONCLUSIONS: Thus, the Noxa/Mcl-1 balance determines sensitivity to ABT-737 in CD40-stimulated CLL cells. These data provide a rationale to investigate the combination of drugs which enhance the Noxa/Mcl-1 balance with ABT-737 to eradicate CLL in chemoresistant niches.

No convincing evidence for a role of CD31-CD38 interactions in the pathogenesis of chronic lymphocytic leukemia.

Although CD38, a marker of poor prognosis in chronic lymphocytic leukemia (CLL), is known primarily as an ecto-enzyme, it has also been ascribed a receptor function. Interaction with its proposed ligand CD31 expressed on nurse-like cells would result in proliferative and survival-signals. Yet, in CLL, both homotypic and heterotypic CD31-CD38 interactions are expected to be rather ubiquitous. We analyzed whether CD38-CD31 interactions result in proliferative and antiapoptotic signals. We found a high expression of CD31 on CLL, irrespective of CD38 expression. Coculture of CD38(high) CLL with endothelial cells or CD31 transfected fibroblasts, with or without blocking CD31 or CD38 antibodies, did not result in increased survival or proliferation. Analysis of gene expression of most known regulators of apoptosis revealed no influence of coculture with CD31-expressing feeder cells. In conclusion, our data do not support an important contribution of CD38 triggering by CD31 to the proliferative and antiapoptotic state of the leukemic clone.