Claire Bonfils

Phase 1 study of the oral isotype specific histone deacetylase inhibitor MGCD0103 in leukemia.

MGCD0103 is an isotype-selective inhibitor of histone deacetylases (HDACs) targeted to isoforms 1, 2, 3, and 11. In a phase 1 study in patients with leukemia or myelodysplastic syndromes (MDS), MGCD0103 was administered orally 3 times weekly without interruption. Twenty-nine patients with a median age of 62 years (range, 32-84 years) were enrolled at planned dose levels (20, 40, and 80 mg/m(2)). The majority of patients (76%) had acute myelogenous leukemia (AML). In all, 24 (83%) of 29 patients had received 1 or more prior chemotherapies (range, 0-5), and 18 (62%) of 29 patients had abnormal cytogenetics. The maximum tolerated dose was determined to be 60 mg/m(2), with dose-limiting toxicities (DLTs) of fatigue, nausea, vomiting, and diarrhea observed at higher doses. Three patients achieved a complete bone marrow response (blasts <or= 5%). Pharmacokinetic analyses indicated absorption of MGCD0103 within 1 hour and an elimination half-life in plasma of 9 (+/- 2) hours. Exposure to MGCD0103 was proportional to dose up to 60 mg/m(2). Analysis of peripheral white cells demonstrated induction of histone acetylation and dose-dependent inhibition of HDAC enzyme activity. In summary, MGCD0103 was safe and had antileukemia activity that was mechanism based in patients with advanced leukemia.

MGCD0103, a novel isotype-selective histone deacetylase inhibitor, has broad spectrum antitumor activity in vitro and in vivo

Nonselective inhibitors of human histone deacetylases (HDAC) are known to have antitumor activity in mice in vivo, and several of them are under clinical investigation. The first of these, Vorinostat (SAHA), has been approved for treatment of cutaneous T-cell lymphoma. Questions remain concerning which HDAC isotype(s) are the best to target for anticancer activity and whether increased efficacy and safety will result with an isotype-selective HDAC inhibitor. We have developed an isotype-selective HDAC inhibitor, MGCD0103, which potently targets human HDAC1 but also has inhibitory activity against HDAC2, HDAC3, and HDAC11 in vitro. In intact cells, MGCD0103 inhibited only a fraction of the total HDAC activity and showed long-lasting inhibitory activity even upon drug removal. MGCD0103 induced hyperacetylation of histones, selectively induced apoptosis, and caused cell cycle blockade in various human cancer cell lines in a dose-dependent manner. MGCD0103 exhibited potent and selective antiproliferative activities against a broad spectrum of human cancer cell lines in vitro, and HDAC inhibitory activity was required for these effects. In vivo, MGCD0103 significantly inhibited growth of human tumor xenografts in nude mice in a dose-dependent manner and the antitumor activity correlated with induction of histone acetylation in tumors. Our findings suggest that the isotype-selective HDAC inhibition by MGCD0103 is sufficient for antitumor activity in vivo and that further clinical investigation is warranted. [Mol Cancer Ther 2008;7(4):759–68]

Phase II study of the histone deacetylase inhibitor MGCD0103 in patients with previously treated chronic lymphocytic leukaemia

MGCD0103, an orally available class I histone deacetylase (HDAC) inhibitor, was examined for pre‐clinical activity in chronic lymphocytic leukaemia (CLL). A phase II clinical trial was performed, starting at a dose of 85 mg/d, three times per week. Dose escalation to 110 mg or the addition of rituximab was permitted in patients without a response after two or more cycles. MGCD0103 demonstrated pre‐clinical activity against CLL cells with a LC50 (concentration lethal to 50%) of 0·23 μmol/l and increased acetylation of the HDAC class I specific target histone H3. Twenty‐one patients received a median of two cycles of MGCD0103 (range, 0–12). All patients had previously received fludarabine, 33% were fludarabine refractory, and 71% had del(11q22·3) or del(17p13·1). No responses according to the National Cancer Institutes 1996 criteria were observed. Three patients received 110 mg and four patients received concomitant rituximab, with no improvement in response. Grade 3–4 toxicity consisted of infections, thrombocytopenia, anaemia, diarrhoea, and fatigue. HDAC inhibition was observed in six out of nine patients on day 8. Limited activity was observed with single agent MGCD0103 in high risk patients with CLL. Future investigations in CLL should focus on broad HDAC inhibition, combination strategies, and approaches to diminish constitutional symptoms associated with this class of drugs.

Expression and characterization of a dictyostelium discoideum annexin

The annexins are calcium-dependent phospholipid-binding proteins. Recently the gene encoding the homologue of a mammalian annexin has been identified inDictyostelium discoideum. Analysis of cDNA and genomic clones showed that the transcript forDictyostelium annexin is alternatively spliced (Greenwood, M. and Tsang, A. (1991) Biochim. Biophys. Acta 1088, 429–432; Döring, V., Schleicher, M and Noegel, A. (1991) J. Biol. Chem. 266, 17509–17515). Here, we showed that theDictyostelium annexin DNA hybridized to two populations of transcripts. We used a recombinant annexin polypeptide to raise polyclonal antibody. Immunoblot analysis revealed that the antibody recognized two polypeptides of 48 kDa and 54 kDa in developingD. discoideum cells. The molecular sizes of these polypeptides correspond well with the expected sizes of the alternatively spliced products. The 48-kDa and 54-kDa polypeptides were purified by isoelectric focusing to more than 70% homogeneity. The partially purified proteins were found to associate with phosphatidylserine vesicles in a calcium-dependent manner. These results suggest that the 48- and 54-kDa polypeptides are the products of alternative splicing of the annexin transcripts. During development the two polypeptides accumulate at different rates to about 60 times the level detected in vegetative cells. On the other hand, RNA blot analysis showed that the level of the annexin transcripts in multicellular aggregates was about 5 times that of vegetative cells.

A prespore-specific gene ofDictyostelium discoideum encodes the small subunit of ribonucleotide reductase

We have isolated the gene. rnrB, that encodes the ribonucleotide reductase small subunit of Dictyostelium discoideum. The deduced amino acid sequence of rnrB exhibits about 60% sequence identity with its homologues in other eukaryotes. As demonstrated by RNA blot analysis the rnrB transcript is detected in growing cells and decreases dramatically at the onset of development. The rnrB transcript reappears after the cells have formed multicellular aggregates. To further examine the pattern of expression, we have fused the rnrB promoter and part of its coding sequence to lacZ. The transgenic strain bearing such a reporter construct expresses the fusion gene with a biphasic profile, which is indistinguishable from that of the endogenous rnrB. The multicellular aggregates of Dictyostelium are differentiated along the anterior-posterior axis. Cells in the anterior give rise to the stalk of the fruiting body while cells in the posterior are precursors of spores. Results from histochemical staining show that beta-galactosidase activity is detected exclusively in the posterior two-thirds of the aggregates. These data suggest that rnrB is expressed in prespore cells during postaggregative development and in vegetative cells.

Identification of cis-regulating elements and trans-acting factors regulating the expression of the gene encoding the small subunit of ribonucleotide reductase in Dictyostelium discoideum.

We have examined the promoter ofrnrB, the gene encoding the small subunit of ribonucleotide reductase of Dictyostelium discoideum, usinglacZ as a reporter gene. Deletion analysis showed that expression of this gene in vegetative cells involves an A/T-rich element, whereas its expression in prespore cells during development requires a region encompassing two G/C-rich elements, designated box A and box B. Removal of boxes A and B results in very low level of activity. When either box A or box B is deleted, prestalk cells adjacent to the prespore zone also express β-galactosidase. The behavior of these cis-regulatory elements implies that the mechanism regulating the prespore-specific expression ofrnrB is different from that regulating other known prespore genes. We have used electrophoretic mobility shift assays to identify factors that interact with box A and box B. Box A interacts with a factor that is found in the nuclear fraction. While box B interacts with a factor that is present in the cytosolic fraction throughout growth and development, its presence in the nuclear fraction is developmentally regulated. Results from competition assays suggest that both box A and box B interact with transcriptional activators that have not been characterized previously.

Abstract 1790: The combination of MGCD265, a Met/VEGFR inhibitor in clinical development, and erlotinib potently inhibits tumor growth by altering multiple pathways including glycolysis

MGCD265 is an oral, multitargeted, receptor tyrosine kinase inhibitor in clinical development. MGCD265 targets the Met, VEGFR1, 2, 3, Tie2 and Ron receptor tyrosine kinases and blocks key pathways regulating cancer development and progression. Met-targeted agents, including MGCD265, have shown early signs of clinical activities when combined with EGFR inhibitors. While this combination is supported by a large body of evidence indicating that Met and EGFR functionally cooperate to amplify activating signals, how the combination of agents targeting these two pathways leads to efficient anti-tumor responses remains to be elucidated. We have previously demonstrated that the combination of MGCD265 with erlotinib achieved greater tumor growth inhibition than treatment with either agent alone in multiple xenografts including a gastric cancer model in which Met is amplified (MKN45). We performed gene expression analyses in this model to gain insight into the molecular mechanisms underlying the efficient anti-tumor activities of MGCD265 in combination with erlotinib. While these studies revealed the inhibition of cell growth and the induction of apoptosis pathways as enhanced by this combination, the regulation of glycolysis surfaced as a novel pathway altered by this combination. Although genes regulating this pathway were partly downmodulated by either agent alone, the combination significantly enhanced this effect. A key mediator of glycolysis, hexokinase 2 (HK2), known to be elevated in cancer cells and essential for the switch to aerobic glycolysis as well as for mitochondrial-initiated cell death, was significantly downregulated by the combination. Furthermore, PFKFB3, PGK, ENO1, PKM2 were also inhibited by the combination. These results suggest that regulating tumor cell glycolysis may present a novel pathway through which MGCD265 could block tumor cell growth. Thus, the inhibition of multiple molecular pathways involved in cancer growth and progression provides a rationale for the clinical development of MGCD265 in combination with erlotinib. Early results from an ongoing Phase I/II clinical trial of MGCD265 in combination with erlotinib has demonstrated encouraging signs of activity in gastric cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1790. doi:1538-7445.AM2012-1790

Pharmacological inhibition of histone deacetylases for the treatment of cancer, neurodegenerative disorders and inflammatory diseases

Background: Histone deacetylases (HDACs) constitute a family of enzymes that deacetylate histones and other cellular proteins. They are major regulators of transcription and are also important in other cellular processes. Objective: The review provides an updated summary of HDAC pharmacological inhibition in clinical oncology, as well as in preclinical studies on inflammation and neurodegenerative diseases. Results/conclusion: HDAC inhibition is a validated approach in cancer therapy, as evidenced by the approval of vorinostat and by encouraging clinical data from various HDAC inhibitors. Moreover, preclinical proof-of-concept studies are emerging from animal models for non-oncologic diseases, including inflammatory and neurodegenerative diseases. The identification of the appropriate target spectrum and the development of class- or isotype-selective inhibitors will be central events in the future.

Abstract 930: Preclinical characterization of MG516, a novel inhibitor of receptor tyrosine kinases involved in resistance to targeted therapies.

Despite breakthroughs in the clinical development of tyrosine kinase inhibitors, challenges remain in overcoming resistance to these molecular targeted therapies. Advances in our understanding of mechanisms of resistance to targeted agents will improve patient outcome. While secondary mutations play a key role, the activation of parallel signaling pathways has been shown to alter the sensitivity to targeted inhibition. Resistance to inhibitors of the EGFR or VEGFR families may occur through the activation of Met, EphA2 and Axl receptor tyrosine kinase pathways, suggesting combined inhibition of these targets as a strategy to prevent resistance to approved EGFR- and VEGFRs -targeted therapies. We have developed a novel multitargeted receptor tyrosine kinase inhibitor, MG516, with nanomolar activities in in vitro enzymatic assays against members of the Eph receptor family, Axl, Met and VEGFR1,2,3. In carcinoma cell lines, MG516 potently inhibits phosphorylation of EphA2, Axl and Met. Inhibition of Met downstream signaling as well as the inhibition of Met-dependent biological endpoints, such as motility and wound healing is also achieved. In human umbilical vein endothelial cells (HUVECs),VEGFR2 activation and VEGF-dependent angiogenesis are blocked. Potent anti-tumor activity is demonstrated across a broad range of human xenograft models including lung, gastric, glioblastoma, colorectal and breast carcinomas. Anti-tumor activity is achieved at oral doses as low as 2.5mg/kg in the absence of overt toxicity, weight loss or myelosuppression. Immunohistochemistry analyses of xenograft tumors after treatment with MG516 reveal a decrease in the proliferation of tumor cells, a decrease in tumor vascularization, pharmacodynamic inhibition of target phosphorylation and decreases in target expression, including EphA2. Consistent with targeting multiple oncogenic pathways simultaneously, the combination of MG516 with EGFR inhibition results in improved tumor growth inhibition. Importantly, in a gastric cancer model exhibiting resistance to sunitinib following prolonged treatment with this agent, MG516 induces tumor regression. Thus, MG516 offers potential for clinical development of a novel therapeutic, by targeting a combination of oncogenic kinases involved in tumor development, progression and resistance to targeted therapies. Citation Format: Normand Beaulieu, Helene Sainte-Croix, Claire Bonfils, Michael Mannion, Stephane Raeppel, Lubo Isakovic, Stephen Claridge, Oscar Saavedra, Franck Raeppel, Arkadii Vaisburg, James Wang, Marielle Fournel, Jeffrey M. Besterman, Christiane R. Maroun. Preclinical characterization of MG516, a novel inhibitor of receptor tyrosine kinases involved in resistance to targeted therapies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 930. doi:10.1158/1538-7445.AM2013-930

Abstract 3612: Potent preclinical anti-tumor activity of MGCD265, an orally active Met/VEGFR multitargeted kinase inhibitor in Phase II clinical development, in combination with an EGFR inhibitor

MGCD265 is an oral multitargeted receptor tyrosine kinase inhibitor in Phase II clinical development. MGCD265 targets the Met receptor tyrosine kinase and blocks Met activities which contribute to cancer development and progression such as cell proliferation, motility/invasion, angiogenesis and tumor cell survival. Although there is a large body of literature supporting Met activities as key to epithelial-mesenchymal transition and tumorigenesis, it is now accepted that the coordination of Met signaling with other regulators is central to oncogenesis. Met engages in cross-talks with several membrane proteins including the EGFR. Met and EGFR are coexpressed on tumor cells and functionally cooperate to amplify activating signals. Moreover, in NSCLC, Met gene amplification or overexpression of HGF, has been identified as a major molecular mechanism through which tumors evade EGFR inhibition by specific inhibitors such as gefitinib and erlotinib. Taken together, these studies provide a compelling rationale for concomitantly inhibiting Met and EGFR. In the present study, we have analyzed the in vivo anti-tumor activity of MGCD265 in combination with an EGFR-specific inhibitor, erlotinib. We demonstrate that this combination achieves greater antitumor responses than treatment with either agent alone, in multiple xenografts including NSCLC models. This is observed in the absence of overt toxicity. Furthermore, the PK of MGCD265 and erlotinib was analyzed when the two agents were co-administered, and indicated that there are no drug-drug interactions. In addition, in a NSCLC xenograft model that expresses an EGFR mutant resistant to erlotinib (T790M), MGCD265 significantly improved the anti-tumor activity when combined with erlotinib. The mechanisms underlying efficient tumor growth inhibition by this combination are presently under investigation. Preliminary results suggest that in addition to downregulating Met activation, MGCD265 may modulate EGFR activation by inhibiting the expression of the EGFR ligands TGFα and EREG. These preclinical studies provide support for the clinical development of MGCD265 in combination with erlotinib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3612.