ALBERT

Description: We have shown that insulin-like growth factors (IGFs) and their receptor IGF-1R play critical roles in proliferation, survival and drug-resistance of a broad spectrum of hematologic malignancies and solid tumors and that selective inhibitors of IGF-1R kinase activity have in vivo anti-tumor activity in clinically relevant orthotopic tumor models (Cancer Cell2004;5:221–30). We now describe the in vitro and in vivo activity of NVP-AEW541, a novel pyrrolo[2,3-d] pyrimidine selective IGF-1R tyrosine kinase inhibitor with 27-fold selectivity for IGF-1R vs. its highly homologous insulin receptor. NVP-AEW541 is active (at sub-uM levels) against diverse tumor types, including 〉30 MM cell lines and 〉10 primary tumor cells from MM patients (including cells resistant to Dex, alkylating agents, anthracyclines, thalidomide, or its immunomodulatory derivatives, IMiDs, bortezomib, and/or Apo2L/TRAIL); and cell lines from diverse hematologic malignancies (including B- and T-ALL, AML, CML, and lymphoma subtypes) and solid tumors (e.g. breast, prostate, lung, thyroid, ovarian, renal Ca, retinoblastoma and sarcomas). All studied tumor cells expressed IGF-1R, without any correlation between mean fluorescence intensity of expression of IGF-1R (or its decoy non-signaling counterpart IGF-2R/CD222) and the degree of tumor cell sensitivity to NVP-AEW541. The in vitro anti-tumor effects of NVP-AEW541 were highly consistent with those of other selective anti-IGF-1R neutralizing agents, including another IGF-IR inhibitor of the pyrrolo[2,3-d] pyrimidine structural class (NVP-ADW742) or anti-human IGF-1R-specific neutralizing mAb’s (aIR3). NVP-AEW541 counteracts the proliferative/anti-apoptotic effect of serum on tumor cells from the entire spectrum of diseases that were studied, but more prominently against MM cells. Importantly, NVP-AEW541 had in vivo anti-tumor activity in a SCID/NOD mice model of diffuse MM. Mechanistically, IGF-1R inhibition by NVP-AEW541 blocks key growth/survival pathways (e.g. PI-3K/Akt, Ras/Raf/MAPK, IKK-a/NF-kB); blocks expression of inhibitors of apoptosis (e.g. FLIP, cIAP-2, survivin); and suppresses both constitutive and serum- or IGF-1-induced upregulation of proteasome activity. These molecular sequelae can explain why NVP-AEW541 sensitized tumor cells (e.g. MM, PrCa, BrCa or sarcomas) to other anti-cancer drugs (e.g. Dex, cytotoxic chemotherapeutics and PS-341); blunted tumor cell responses to other growth factors (e.g. MM or PrCa cell response to IL-6); overcame the drug-resistance phenotype conferred by bone marrow stromal cells; and abrogated VEGF production in co-cultures of MM cells with BMSCs. These studies further confirm that IGF-1R plays major role in growth/survival of neoplastic cells, indicate that IGF-1R pathway can be targeted with multiple clinically applicable approaches; and provide proof-of-principle for clinical trials of NVP-AEW541, e.g. in MM, a disease particularly dependent upon IGF-1R function.

Description: Multiple myeloma (MM) remains an incurable neoplasia, despite recent development of several novel therapies. As part of our efforts to identify new compounds with anti-MM activity, we evaluated the class of avicins, which are triterpenoid saponins that have been shown to induce apoptosis of neoplastic cells by affecting mitochondrial function independently of membrane-bound death receptors. Because we have previously shown that mitochondria constitute key regulators of MM cell responsiveness to diverse anti-tumor agents, (e.g. the proteasome inhibitor bortezomib), we evaluated the in vitro anti-MM effects of this class of compounds. Our vitro drug-sensitivity studies showed that Avicin D and Avicin G, the main members of this class of compounds, are active against a broad panel of MM cell lines and primary tumor cells, including tumor cells resistant to conventional (e.g. dexamethasone, alkylating agents, anthracyclines) or novel (e.g. thalidomide, immunomodulatory thalidomide derivatives, proteasome inhibitor PS-341[bortezomib], Apo2L/TRAIL) anti-MM agents. Using MTT survival assays, we confirmed that the IC50 values for both Avicins were highly concordant and were less than 250 nM for the overwhelming majority of MM cell lines tested. Importantly, this potent in vitro anti-MM activity was triggered by concentrations of Avicins which had minimal, if any, effect on the viability of normal hematopoietic cells or bone marrow stromal cells. Furthermore these IC50 values were comparable with the in vitro activity of this agent among the most Avicin-sensitive tumor models that have been previously tested. This potent anti-MM effect was not inhibited by transfection of MM cells with construct for constitutively active Akt. Although cytokine- or cell adhesion-mediated interactions of the local bone marrow (BM) microenvironment (e.g. BM stromal cells) protects MM cells from conventional therapies (e.g. dexamethasone or cytotoxic chemotherapy), avicins were able to overcome this protective effect in co-culture models of MM cells with BM stromal cells and sensitized MM cells to cytotoxic chemotherapy-induced cell death. Using hierarchical clustering analyses and relevance network algorithms, we compared the patterns of MM cell sensitivity to Avicin D vs Avicin G vs. other anti-cancer drugs and found that the pattern of dose-response relationships of the 2 main members of this class of compounds are very similar to each other, but clearly distinct from the patterns of sensitivity or resistance to other drugs, either conventional or investigational. This further supports the notion that the anti-MM properties of Avicins are mediated by molecular mechanisms distinct from those of currently available anti-MM drugs, and also suggests that Avicins may have anti-tumor activity even against subgroups of MM which may be resistant to other novel therapies that are currently in clinical development. These results have provided the framework for ongoing in vivo studies of anti-tumor activity of these agents, to evaluate the feasibility of future clinical trials of Avicins to improve patient outcome in MM.

Description: Introduction Plitidepsin is a cyclic depsipeptide isolated from the marine tunicate, Aplidium albicans with promising antitumor activity. This work represents a comprehensive study (in vitro, in vivo and clinical) of its antimyeloma efficacy. Material & Methods In vitro studies were performed in 23 multiple myeloma (MM) cell lines and in cells from 16 MM patients. For the in vivo analysis a human plasmocytoma model in CB17-SCID mouse was used. Mice were randomized to receive Aplidin® 100 μg/Kg ip x 7 days/week (n=9), Aplidin® 140 μg/Kg ip x 5 days/week (n=7) or vehicle alone (n=9). The clinical efficacy of Aplidin® in relapsed/refractory patients was evaluated in a non-randomized two-stage Phase II, multicenter, clinical trial. Dosage of Aplidin® was 5 mg/m2 every 2 weeks. Results Aplidin® showed clear in vitro efficacy (IC50:1–10 nM) in the 23 cell lines tested including those resistant to dexamethasone, melphalan or doxorubicin. It was also active in the presence of microenvironment (IL-6, IGF-1 and BMSCs). Thirteen out of the 16 patient samples were sensitive to Aplidin® with 〉80% cell death in 8 cases and 60–80% in the remaining ones without significant toxicity in non tumor cells. Combination of Aplidin® with dexamethasone, bortezomib or lenalidomide showed clear potentiation. Aplidin® acts by inducing apoptosis with caspase−3, −7, −8, −9 and PARP cleavage. It also involves the activation of p38 and JNK signalling, Fas/CD95 translocation to lipid rafts and downregulation of Mcl-1 and myc. In mice studies, both schedules of treatment reduced tumor growth and increased survival with statistical differences in the group receiving 140 μg/Kg x 5d/week (p=0.04, Log Rank p=0.02). No significant toxicity was observed. These data provided the rationale for a clinical trial that has included 31 patients with relapsed/refractory MM. Median age was 65 years (47–82) and the median number of prior lines of therapy was 4 (range: 1–9) including autologous stem cell transplant (60%), thalidomide (58%) and bortezomib (48%). Out of the 26 evaluable patients, 2 (8%) achieved PR and 3 (12%) MR. Eight patients (31%) remained in stable disease (SD). Due to the synergism with dexamethasone observed in the in vitro studies, the protocol was amended to allow the addition of this agent in pts progressing after 3 cycles or with SD after 4 cycles. With a median follow-up of 14 months (range: 6.8–16.3), the time to progression in responding pts was 5.8 months (4.9–7.6). The most common G3-4 adverse events were fatigue (7%), serum creatine phosphokinase increase (7%), muscle toxicity (10%) and hepatic toxicity (10%). No significant hematologic toxicity or neuropathy was observed. Conclusion Aplidin® is effective both as a single agent and in combination with dexamethasone in the in vitro and in vivo settings. Its activity in relapsed/refractory MM patients is promising with an acceptable toxicity profile.

Description: The Ras/Raf/MEK/MAPK pathway is a major regulator of tumor cell proliferation. Ras mutations have been known in many malignancies for several years, whereas more recent reports have shown that activating mutations of the BRAF gene are present in a large percentage of human malignant melanomas and thyroid carcinomas and in a substantial proportion of colon carcinomas (Nature2002;417:949–54). The vast majority of these mutations represent a T1796A change resulting in a V599E substitution within the activation segment of B-Raf. This mutant B-Raf kinase is constitutively active and results in inappropriate stimulation of downstream MAPK/ERKs and tumor cell proliferation. The PTPN11 gene encodes SHP-2 (Src homology 2 domain-containing protein tyrosine Phosphatase), a nonreceptor tyrosine protein tyrosine phosphatase (PTPase) that modulates Ras signaling. Germline mutations in PTPN11 are responsible for Noonan syndrome (NS), whereas somatic mutations have been detected in juvenile myelomonocytic leukemia (JMML). We investigated the presence of mutations in the Ras/B-Raf/SHP-2 axis in 25 lines from B-cell malignancies, including 19 multiple myeloma (MM) cell lines. DNA was isolated and B-Raf (exons 11 and 15), PTPN11 (3 and 13) and K-, N- and H-Ras sequences were amplified by PCR using specific flanking primers. The PCR products were purified and sequenced in automatic sequencer (ABI PRISM 3100 Genetic Analyzer; Applied Biosystems). Ras gene mutations were found in 11/25 (44%) of the studied cell lines. None of the previously described activating B-Raf mutations, including the most prevalent V599E, were found in our panel. Only one MM cell line demonstrated a C1332A substitution, which corresponds to a D444E change, which is predicted to have a minimal, if any, impact on kinase activity. A single PTPN11 G178C substitution, corresponding to a G60R change (previously described in NS and AML), was detected in another MM line. These data confirm the previously known role of Ras mutations in B-cell malignancies and identify a rare occurrence of a PTPN11 mutation in our panel. On the other hand, activating mutations of the B-Raf kinase are unlikely to play a significant part in the pathogenesis of B cell malignancies, in contrast to their prominent role in several types of solid tumors. In total, our data suggest that the Ras/Raf/MEK/MAPK can be overactive in these malignancies due to activating genetic events upstream of B-Raf, such as the previously described activating Ras mutations.