Abstract 1331
BET (bromodomain and extra-terminal) proteins bind to acetylated lysine residues on histones and thereby directly and selectively regulate the expression of genes relevant to cancer. ...Small molecule inhibition of BET protein binding to chromatin suppresses the transcription of MYC, a subset of NF-κB-dependent genes, and BCL-2. Because of the clinical potential of this novel mechanism, we have discovered selective and potent small molecule inhibitors of BET bromodomains with physical and pharmacokinetic properties that are favorable for clinical development. CPI-267232 is representative of a series of small molecule BET inhibitors with these characteristics. In a biochemical binding assay it has an IC50 126 nM against BD1 of BRD4, and in the MV4-11 leukemia cell line it suppresses the transcription of MYC with an EC50 of 170 nM and inhibits its growth with a GI50of 120 nM. In a panel of more than 100 cancer cell lines CPI-267232 and other structurally dissimilar BET inhibitors demonstrate growth inhibitory activity most potently and consistently against cell lines of hematologic origin. Cells treated with CPI-267232 undergo a G1 arrest with the more sensitive lines undergoing apoptosis with longer periods of drug exposure (> 48 hrs). CPI-267232 has high oral bioavailability in mice (∼80%) but is cleared rapidly, with an elimination half-life of approximately 3 hrs (in dogs, oral bioavailability is 94% with an elimination half-life of 9 hrs). PK-PD studies conducted in mice bearing subcutaneous xenografts of Raji Burkitt’s lymphoma demonstrated that maximal (80%) suppression of MYC expression was achieved 4 hours following a single 30 mg/kg oral dose; MYC expression returned to baseline levels by 12 hours, consistent with the rapid elimination of the compound. Efficacy studies in nude mice bearing xenografts were subsequently conducted, with a 30 mg/kg PO bid regimen yielding a %T/C value of 23% (Raji) or regression (MV4-11) after 21 days of treatment. Evaluation of the relationships between various measurements of drug exposure and efficacy revealed that efficacy is primarily driven by maintaining drug concentration above a minimum value rather by total AUC or Cmax. This observation is consistent with the importance of exposure duration in effecting growth arrest and cell killing in tissue culture. Although the transcription of MYC is frequently suppressed, the overall effects of BET inhibition on gene transcription vary across cell lines of different origin. Genome-wide expression profiling of 2 myeloma and 3 leukemia cells lines at 4 and 24 hours after exposure to a small molecule BET inhibitor resulted in the identification of approximately 200 genes that were commonly either up- or down-regulated by at least 2-fold with statistical significance. In addition, a small set of BET inhibitor-sensitive genes has been identified in peripheral blood mononuclear cells. The discovery of novel BET inhibitors with optimized potency, selectivity, and pharmacokinetic properties, coupled with these insights into the pharmacokinetic and pharmacodynamic determinants of anti-tumor activity, provides an opportunity for the rational development of BET inhibition in the treatment of patients with hematologic malignancies.
Sims:Constellation Pharmaceuticals: Employment. Normant:Constellation Pharmaceuticals: Employment. Sandy:Constellation Pharmaceuticals: Employment. Mertz:Constellation Pharmaceuticals: Employment. Bryant:Constellation Pharmaceuticals: Employment. O'Meara:Constellation Pharmaceuticals: Employment. Green:Constellation Pharmaceuticals: Employment. Cooper:Constellation Pharmaceuticals: Employment. Audia:Constellation Pharmaceuticals: Employment.
Chronic myeloid leukaemia (CML) is a hematological malignancy resulting from the transformation of a primitive hematopoietic progenitor by the fusion oncogene BCR-ABL, a constitutively active ...tyrosine kinase. In recent years major advances have been made in the treatment of CML with the development of tyrosine kinase inhibitors (TKIs), resulting in high rates of remission in CML chronic phase (CP) patients. However, relapse is driven by quiescent and self-renewing BCR-ABL+ CML stem cells (LSCs) that are resistant to TKIs. Consequently, identification of novel proteins or pathways which can be drug-targeted to eliminate the LSCs is a primary goal of current CML research.
Through comparative analysis between CML and non-leukemic samples, we show that components of the repressive Polycomb group (PcG) complex PRC2 are significantly misregulated in CML samples. By performing genome-wide mRNA and epigenetic screens, we demonstrate that this has led to as many as 3-fold more gene repression events in CML cells being associated with gains in the histone modification H3K27me3. This misregulation results in different biological pathways being targeted by PRC2 than those found in non-leukemic samples. We demonstrate that the majority of this misregulation is present in the LSCs.
EZH2 is a key component of the PRC2 complex, responsible for laying down the H3K27me3 mark. To determine the effect of inhibition of the complex on LSC survival we have utilised an inhibitor of EZH2, CPI-625. In the absence and presence of TKI, treatment of CP CML CD34+ cells (n=3) with CPI-625 resulted in decreased cell viability (p<0.001 and p<0.05, -/+ TKI respectively) and increased apoptosis (p<0.05 without TKI) in a dose dependent manner. Significantly, there was also a decrease in the number of cells in the undivided, quiescent ‘TKI resistant' population relative to controls (p<0.01 and p<0.05 -/+ TKI respectively). This was accompanied by an increase in apoptosis (p<0.05 without TKI). Moreover, treatment with CPI-625 resulted in decreasing Colony Forming Cell (CFC) numbers, both in the absence (p<0.05) and presence (p<0.01) of TKI relative to controls. Similar results were seen with treatment of the more primitive CD34+38- cells. Importantly, these effects were not observed in non-leukemic cells. These results demonstrate that CPI-625 is capable of selective targeting of the LSC population.
Our data strongly points to changes in H3K27me3 gene targets in CML as a feature related to misregulation of the PRC2 complex. We have demonstrated that targeting of this complex may have efficacy in the treatment of CML, including eradication of the drug resistant LSCs.
Holyoake:Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees.
Abstract
Histone lysine methylation, which is dynamically regulated by methyltransferases and demethylases, plays an important role in the establishment and maintenance of chromatin structure and ...thereby contributes to the control of gene expression. The development of small molecule methyltransferase and demethylase inhibitors provides an approach to manipulate transcriptional programs, and thus potentially allow interference with aberrant cellular states as observed in cancer.
The histone methyltransferase Enhancer of Zeste Homologue 2 (EZH2) is a component of the Polycomb Repressive Complex 2. It mediates trimethylation of lysine 27 on histone 3 (H3K27me3), which leads to transcriptional repression. EZH2 has been widely implicated in cancer and inhibition of its catalytic activity provides a novel therapeutic approach to treat human cancers. Constellation has identified, characterized and optimized potent, selective and reversible EZH2 small molecule inhibitors as well as studied the biological impact of such inhibition. We have previously shown that pharmacological inhibition of EZH2 causes selective lymphoma cell viability defects with cell lines harboring EZH2 mutations being the most sensitive.
The discovery of small molecules that specifically inhibit EZH2 has enabled us to look for other disease indications that might be dependent on EZH2 for survival. We carried out a long term cell viability screen in ∼75 cell lines across several different hematological malignancies using an EZH2 inhibitor. About 30% of all tested Mutliple Myeloma and Plasmacytoma cell lines showed a time-dependent phenotypic response. In these cell lines H3K27me3 levels were effectively reduced in a dose dependent manner within 4 days of compound treatment which was followed by the induction of apoptotsis at later time points. EZH2 inhibitors also achieved tumor growth inhibition in a Multiple Myeloma subcutaneous xenograft model. To understand the underlying molecular mechanism of EZH2 inhibitor sensitivity in Multiple Myeloma, genome-wide mapping of EZH2 and H3K27me3 sites in the absence and presence of the compound have been performed in conjunction with gene expression profiling and the results will be discussed. In conclusion, we identified Multiple Myeloma as a disease modality where EZH2 inhibition leads to cell viability defects both in vitro and in vivo.
Citation Format: Shilpi Arora, Vidya Balasubramanian, Kaylyn Williamson, Victor Gehling, Chris Nasveschuk, Rishi Vaswani, Jennifer Busby, Shivani Garapaty, Priya Iyer, Feng Zhao, Robert Campbell, Richard Cummings, Jim Audia, JC Harmange, Brian Albrecht, Andrew Cook, Les Dakin, Emmanuel Normant, Patrick Trojer. Inhibition of the histone methyl transferase EZH2 causes viability defects in multiple myeloma. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5526. doi:10.1158/1538-7445.AM2014-5526
Abstract
Heat shock protein 90 (Hsp90) plays a role in regulating the stability of key cancer-causing proteins through its role as a protein chaperone. Proteins chaperoned by Hsp90, known as client ...proteins, include cancer-causing forms of ALK, BCR-ABL, EGFR, FLT3 and HER2. Infinity is developing two drug candidates in its Hsp90 chaperone inhibitor program: IPI-504 (retaspimycin hydrochloride), an intravenously-administered small molecule, and IPI-493, which is administered orally.
EGFR tyrosine kinase inhibitors (TKIs) are an effective treatment for lung cancer patients with activating mutations in EGFR. After a dramatic initial response, however, most patients become resistant to drug treatment and progress. In about half of these cases, resistance is due to a second point mutation in EGFR (T790M). It is believed that in at least some of these cases, the TKI resistance mutations are pre-existing and that treatment with TKIs selects for the resistant cells.
In an effort to model the emergence of resistance to TKIs from pre-existing mutations, we developed a novel in vivo model, where gefitinib treatment initially leads to tumor regression followed by rebound of tumor growth and outgrowth of drug resistant clones containing the T790M mutation. We show that in this model, treatment with IPI-493 alone and IPI-493 following gefitinib resulted in tumor growth inhibition of 61 and 77%, respectively, when compared with gefitinib treatment alone. Treatment with IPI-493 alone also resulted in a significant delay in time to tumor progression with ∼40% of animals still on study 45 days following tumor implant; all animals treated with either vehicle or gefitinib had been removed due to tumor progression. Interestingly, treatment with IPI-493 following gefitinib resulted in an even more impressive delay in time to progression, with >50% of animals still on study on day 65 post-implant.
These results suggest that further studies with Hsp90 inhibitors in EGFR mutant NSCLC patients who have been pre-treated with a TKI may be warranted.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1601. doi:10.1158/1538-7445.AM2011-1601
Abstract
Heat shock protein 90 (Hsp90) plays a role in regulating the stability of key cancer-causing proteins through its role as a protein chaperone. Proteins chaperoned by Hsp90, known as client ...proteins, include cancer-causing forms of ALK, BCR-ABL, EGFR, FLT3, and HER2. Infinity is developing two drug candidates in its Hsp90 chaperone inhibitor program: IPI-504 (retaspimycin hydrochloride), an intravenously-administered small molecule, and IPI-493, which is administered orally. To investigate the activity of IPI-493 in colorectal cancer (CRC), we performed in vitro growth inhibition (GI) studies on a CRC cell line panel. IPI-493 demonstrated GI50s in the range of 10-100nM in cell lines harboring either kRAS or bRAF mutations. To explore the in vivo potency of IPI-493, several mutant bRAF, mutant kRAS, as well as a wild-type kRAS/bRAF xenograft models were developed. Administration of IPI-493 at 100 mg/kg three times weekly demonstrated dramatic effects in all of the mutant models tested, with tumor growth inhibition (TGI) values between 70 and 90% and regression in one mutant bRAF model. Importantly, when IPI-493 was evaluated in combination with irinotecan, the combination resulted in greater tumor regression than seen with either agent alone with complete regressions observed in 4 of 10 animals. Interestingly, in the wild-type kRAS/bRAF models, IPI-493 administration did not lead to tumor growth inhibition. These results suggest that activation of the MAPK pathway may predispose these cells to sensitivity to Hsp90 inhibition. To investigate the effect of IPI-493 on MAPK pathway activity, we performed pharmacodynamic analysis after a single dose of IPI-493 in multiple xenograft models differing in their RAF/RAS mutation status. In mutant bRAF models, pathway activity was high, and IPI-493 administration resulted in downregulation of the activity of both bRAF and MEK. In models containing no mutations in kRAS or bRAF, we detect low baseline levels of both p-bRAF and p-MEK and little effect of IPI-493 administration. When Ras pathway activity in all CRC xenografts was compared with IPI-493 efficacy, there was a clear correlation between pathway activation and tumor growth inhibition by IPI-493. Our finding that Ras pathway activation predisposes CRC cells to sensitivity to IPI-493 and our combination data with irinotecan provide a clear rationale for the evaluation of Hsp90 inhibitors in colorectal cancer.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2827. doi:10.1158/1538-7445.AM2011-2827
The presence of carboxypeptidase A (EC 3.4.17.1; CPA) gene transcripts and corresponding catalytic activity was investigated in brain and other extradigestive rat tissues in which presence of the ...pancreatic enzyme had not been reported so far.
Transcripts of two known CPA genes, CPA1 and CPA2, were identified in extremely low abundance in brain and several other extrapancreatic tissues using Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Whereas the CPA1 gene transcripts in brain, heart, stomach, or colon had a size similar to that in pancreas (1.35 kilobases), the CPA2 gene transcripts in brain, testis, or lung were of a smaller size (1.1 kilobases). Northern blot analysis using various probes, RT-PCR, and 5′-rapid amplification of cDNA 5′-end (5′ RACE analysis) all indicated that this smaller size of the brain transcript was attributable to production by alternative splicing of the pro-mRNA. This process corresponds to deletion of the first four exons, leading to a mRNA encoding a protein in which the signal peptide and activation peptide of prepro-CPA2 are absent but the active site remains. The prediction that the shorter CPA2 isoform, designated CPA2(S), should correspond to a cytoplasmic metallopeptidase that does not require tryptic activation was verified by characterization of the recombinant protein and comparing it with the native CPA-like activity in brain. Both recombinant CPA2(S) generated in Escherichia coli and a soluble protein from brain displayed similar sizes on Western blots (32 kDa to be compared to 34 kDa for pancreatic CPA2). Recombinant CPA2(S) and a soluble CPA-like activity from brain displayed similar sensitivity to a series of inhibitors, contrasting with that of the pancreatic enzyme. It is concluded that alternative splicing produces a truncated CPA2 with distinct subcellular localization and modified catalytic activity.
In spite of the presence of the CPA1 mRNA, no corresponding CPA activity could be detected in brain extracts, even after tryptic activation. This apparent discrepancy seems attributable to the presence of an endogenous peptide inhibitor which remains to be identified.
Abstract
A phase 1 study of IPI-504 (retaspimycin hydrochloride) administered intravenously twice weekly for 2 weeks at 22.5, 45, 90, 150, 225, 300 or 400 mg/m2 followed by 10 days off-treatment was ...conducted to determine the safety and maximum tolerated dose (MTD) of IPI-504 in patients with relapsed or relapsed/refractory multiple myeloma (MM). Anti-tumor activity and pharmacokinetics were also evaluated. Eighteen patients (mean age 60.5 years; median 9 prior therapies) were enrolled. No dose-limiting toxicities (DLTs) were reported for IPI-504 doses up to 400 mg/m2. The most common treatment-related adverse event was grade 1 infusion site pain (four patients). All other treatment-related events were assessed as grade 1 or 2 in severity. The area under the curve (AUC) increased with increasing dose, and the mean half-life was approximately 2-4 h for IPI-504 and its metabolites. Four patients had stable disease, demonstrating modest single-agent activity in relapsed or relapsed/refractory MM.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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