Histone deacetylase inhibition in combination with MEK or BCL-2 inhibition in multiple myeloma.

Despite recent advances in the treatment of multiple myeloma, patients with this disease still inevitably relapse and become refractory to existing therapies. Mutations in K-RAS, N-RAS and B-RAF are common in multiple myeloma, affecting 50% of patients at diagnosis and >70% at relapse. However, targeting mutated RAS/RAF via MEK inhibition is merely cytostatic in myeloma and largely ineffective in the clinic. We examined mechanisms mediating this resistance and identified histone deacetylase inhibitors as potent synergistic partners. Combining the MEK inhibitor AZD6244 (selumetinib) with the pan-histone deacetylase inhibitor LBH589 (panobinostat) induced synergistic apoptosis in RAS/RAF mutated multiple myeloma cell lines. Interestingly, this synergy was dependent on the pro-apoptotic protein BIM. We determined that while single-agent MEK inhibition increased BIM levels, the protein remained sequestered by antiapoptotic BCL-2 family members. LBH589 dissociated BIM from MCL-1 and BCL-XL, which allowed it to bind BAX/BAK and thereby initiate apoptosis. The AZD6244/LBH589 combination was specifically active in cell lines with more BIM:MCL-1 complexes at baseline; resistant cell lines had more BIM:BCL-2 complexes. Those resistant cell lines were synergistically killed by combining the BH3 mimetic ABT-199 (venetoclax) with LBH589. Using more specific histone deacetylase inhibitors, i.e. MS275 (entinostat) and FK228 (romidepsin), and genetic methods, we determined that concomitant inhibition of histone deacetylases 1 and 2 was sufficient to synergize with either MEK or BCL-2 inhibition. Furthermore, these drug combinations effectively killed plasma cells from myeloma patients ex vivo Given the preponderance of RAS/RAF mutations, and the fact that ABT-199 has demonstrated clinical efficacy in relapsed/refractory multiple myeloma, these drug combinations hold prom ise as biomarker-driven therapies.

Sorafenib, a multikinase inhibitor, is effective in vitro against non-Hodgkin lymphoma and synergizes with the mTOR inhibitor rapamycin.

Non-Hodgkin lymphoma (NHL) represents a heterogenous group of neoplasias originating from lymphoid cells. Increased angiogenesis and expression of Vascular Endothelial Growth Factor (VEGF) and its receptors (VEGFR) have been found to be associated with NHL disease progression. Increase in VEGF and other cytokines stimulate signaling cascades, including the Ras/Raf/Mek/Erk pathway, resulting in increased proliferation and decreased apoptosis. Here, we report the in vitro antilymphoma activity of sorafenib, an inhibitor of VEGFR and Raf kinase. Sorafenib induced potent cytotoxicity in NHL cell lines and patient samples. This induction of cytotoxicity was associated with a corresponding increase in apoptotic cell death. Mechanism of action of sorafenib was investigated in follicular (DoHH2) and Burkitt lymphoma (Raji) cell lines. pStat3, pAkt, Mcl1, and Xiap were downregulated in both cell lines, whereas pErk decreased in Raji but not in DoHH2 cells following sorafenib treatment. IL6 was unable to prevent sorafenib induced repression of pStat3, pAkt, Mcl1, and Bcl-Xl. Sorafenib in combination with an mTORC1 inhibitor rapamycin demonstrated synergy in inducing cytotoxicity in NHL cells. Sorafenib/rapamycin combination resulted in downregulation of pAkt, pmTOR, p-p70S6K, p4EBP1, pGSK3ß, Mcl1, and Bcl-Xl. On the basis of our results, a clinical trial is underway using sorafenib with everolimus in NHL patients.

R-(-)-gossypol (AT-101) activates programmed cell death in multiple myeloma cells.

OBJECTIVE: Bcl-2 family proteins play a critical role in malignancies by regulating the balance between cell survival and apoptosis. R-(-)-gossypol (AT-101) is a small molecule that mimics the BH3 domain of cellular Bcl-2 inhibitors and interferes with the function of prosurvival Bcl-2 proteins. We examined the cytotoxicity of AT-101 in the context of multiple myeloma, a fatal hematological malignancy. MATERIALS AND METHODS: Multiple myeloma cell lines and primary cells obtained from multiple myeloma patients were used to investigate the effects of AT-101. Cell viability, apoptosis, and apoptosis pathways were examined using conventional viability assays, flow cytometry, and immunoblots. RESULTS: AT-101 was not only cytotoxic to conventional multiple myeloma cell lines, but was also effective against drug-resistant cell lines and primary multiple myeloma patient cells. Furthermore, AT-101 decreased proliferation of multiple myeloma cell lines in the presence of marrow stromal cells, indicating that this drug may overcome the protective effect of the microenvironment that is important for multiple myeloma cell proliferation and survival. Apoptosis was activated via the mitochondrial pathway in multiple myeloma cell lines treated with AT-101 as demonstrated by an increased Bax to Bcl-2 ratio, mitochondrial membrane depolarization, and caspase activation. Finally, our studies demonstrated that AT-101 exhibits potent synergy with dexamethasone, a valuable therapeutic for multiple myeloma. CONCLUSION: These data suggest that the activity of AT-101 may be highly relevant to multiple myeloma disease biology and may represent an option for treatment of patients with this disease.

FISH is superior to PCR in detecting t(14;18)(q32;q21)-IgH/bcl-2 in follicular lymphoma using paraffin-embedded tissue samples.

Detection of t(14;18)(q32;q21)-IgH/bcl-2, which is present in 70% to 95% of follicular lymphomas (FLs), might aid in diagnosing FL. The efficacy of routine polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques in detecting t(14;18) in paraffin-embedded tissue samples was compared on 5 normal tonsils and 28 FLs demonstrated to be t(14;18)+ by previous karyotyping. There was technical failure in 14 (50%) of the FLs by PCR, likely due to B-5 fixation, and 4 (14%) of FLs by FISH, likely due to advanced specimen age. In the remaining successful cases, 5 (36%) of 14 were positive by PCR and 24 (100%) of 24 were positive by FISH. All 5 normal tonsils were negative by both methods. FISH is superior to PCR for detecting t(14;18) from paraffin-embedded tissue samples because it is more sensitive and equally specific.

Flt-3 and c-kit mutation studies in a spectrum of chronic myeloid disorders including systemic mast cell disease.

We screened 115 patients with chronic myeloid disorders (CMD) for known flt-3 and c-kit mutations in both the juxtamembrane (JM) and the activation loop (AL) domains. None of the patients displayed flt-3 (JM or AL) or c-kit JM mutations. However, the c-kit AL (D816V) mutation was detected in 5 of 16 patients with systemic mast cell disease (SMCD) but in none of the remaining 99 patients with other CMD. In SMCD, the presence of D816V mutation was significantly associated with advanced age, an aggressive clinical course, increased bone marrow mast cell content, and chronic myelomonocytic leukemia.

Phase II study of bevacizumab in combination with sorafenib in recurrent glioblastoma (N0776): a north central cancer treatment group trial.

PURPOSE: We hypothesized that vertical blockade of VEGF signaling by combining bevacizumab with sorafenib in patients with recurrent glioblastoma would result in a synergistic therapeutic effect. We also investigated whether VEGF, VEGFR2 and hypoxia-inducible factor-1α single-nucleotide polymorphisms (SNP), circulating biomarkers of angiogenesis, and MRI markers such as apparent diffusion coefficient (ADC) are correlated with treatment efficacy and/or toxicity. EXPERIMENTAL DESIGN: Patients received bevacizumab (5 mg/kg every 2 weeks) with sorafenib (200 mg twice a day, weekly, days 1-5; group A). Due to toxicity, the starting sorafenib dose was subsequently modified to 200 mg every day (group B). RESULTS: Fifty-four patients were enrolled: 19 patients in group A and 35 in group B. Objective response rate was 18.5% with median duration of 6.7 months (range 0.5-24.1 months). Six-month progression-free survival (PFS6) was 20.4% (11/54), and median overall survival (OS) was 5.6 months [95% confidence interval (CI), 4.7-8.2]; outcome was similar between the two dose groups. We identified SNPs in the VEGF and VEGFR2 promoter regions, which were associated with PFS6 (P<0.022). Among molecular markers of angiogenesis, a higher log2 baseline level of stromal cell-derived factor-1 was associated with PFS6 success (P=0.04). Circulating endothelial cells decreased during treatment with subsequent increase at disease progression (P=0.022). Imaging analysis showed a trend associating ADC-L with poor outcome. CONCLUSIONS: The bevacizumab/sorafenib combination did not improve outcome of patients with recurrent glioblastoma versus historic bevacizumab-treated controls. Biologic markers of response and resistance to bevacizumab in gliomas were identified which merit prospective validation.

Differential expression of CD2 on neoplastic mast cells in patients with systemic mast cell disease with and without an associated clonal haematological disorder.

Recently, aberrant coexpression of CD2 and CD25 has been reported to reliably distinguish neoplastic mast cells from normal or so-called reactive mast cells. Such expression is included in the consensus diagnostic criteria for systemic mast cell disease (SMCD). In our study of patients with SMCD, we found CD2 expression to be more prevalent on mast cells from patients without an associated haematological disorder (P = 0.04). Furthermore, no correlation was found between mast cell CD2 expression and other clinicopathological features in these patients.

Expression of CD52 on plasma cells in plasma cell proliferative disorders.

Multiple myeloma (MM) and primary systemic amyloidosis (AL) remain incurable disorders, and new treatments targeted to the malignant plasma cells are needed. Alemtuzumab is a humanized monoclonal antibody to CD52 and has activity in chronic lymphocytic leukemia. We examined the CD52 expression on CD45+ and CD45- plasma cell populations to evaluate the potential for using alemtuzumab for these disorders. Bone marrows from 61 patients (29 AL, 23 MM, and 9 MGUS [monoclonal gammopathies of undetermined significance]) were studied using 3-color (CD38/45/52) flow cytometry. Among those with MGUS, MM, and AL, 67%, 52%, and 35%, respectively, were positive for CD52 expression. The CD52 expression was predominantly confined to the clonal CD38+/CD45+ plasma cell fraction with median expression of 68%, 88%, and 82% in MGUS, MM, and AL, respectively, compared with 18%, 6%, and 9% among the CD45- plasma cell population. Clinical trials are warranted in these diseases to learn the therapeutic benefit of anti-CD52 immunotherapy.

Bone marrow angiogenic ability and expression of angiogenic cytokines in myeloma: evidence favoring loss of marrow angiogenesis inhibitory activity with disease progression.

We compared the angiogenic potential of bone marrow plasma and the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and their receptors on plasma cells from patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and newly diagnosed multiple myeloma (NMM). Cytokine and cytokine-receptor expression was studied by bone marrow immunohistochemistry, quantitative reverse transcription-polymerase chain reaction (RT-PCR) on sorted plasma cells, and quantitative Western blot analysis. Bone marrow angiogenic potential was studied using a human in vitro angiogenesis assay. The expression levels of VEGF, bFGF, and their receptors were similar among MGUS, SMM, and NMM. Sixty-one percent of NMM samples stimulated angiogenesis in the in vitro angiogenesis assay compared with SMM (0%) and MGUS (7%) (P <.001). Importantly, 63% of MGUS samples inhibited angiogenesis compared with SMM (43%) and NMM (4%) (P <.001). The inhibitory activity was heat stable, not overcome by the addition of VEGF, and corresponded to a molecular weight below 10 kd by size-exclusion chromatography. Our results suggest that increasing angiogenesis from MGUS to NMM is, at least in part, explained by increasing tumor burden rather than increased expression of VEGF/bFGF by individual plasma cells. The active inhibition of angiogenesis in MGUS is lost with progression, and the angiogenic switch from MGUS to NMM may involve a loss of inhibitory activity.