Christopher Sean Straub

A Smac Mimetic Rescue Screen Reveals Roles for Inhibitor of Apoptosis Proteins in Tumor Necrosis Factor-α Signaling

Smac mimetic compounds targeting the inhibitor of apoptosis proteins (IAP) baculoviral IAP repeat-3 domain are presumed to reduce the threshold for apoptotic cell death by alleviating caspase-9 repression. We explored this tenet in an unbiased manner by searching for small interfering RNAs that are able to confer resistance to the Smac mimetic compound LBW242. Among the screening hits were multiple components of the tumor necrosis factor alpha (TNFalpha) signaling pathway as well as X-linked inhibitor of apoptosis (XIAP) itself. Here, we show that in a subset of highly sensitive tumor cell lines, activity of LBW242 is dependent on TNFalpha signaling. Mechanistic studies indicate that in this context, XIAP is a positive modulator of TNFalpha induction whereas cellular inhibitor of apoptosis protein 1 negatively regulates TNFalpha-mediated apoptosis.

IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism.

Oncogenic mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in several types of cancer, but the metabolic consequences of these genetic changes are not fully understood. In this study, we performed (13)C metabolic flux analysis on a panel of isogenic cell lines containing heterozygous IDH1/2 mutations. We observed that under hypoxic conditions, IDH1-mutant cells exhibited increased oxidative tricarboxylic acid metabolism along with decreased reductive glutamine metabolism, but not IDH2-mutant cells. However, selective inhibition of mutant IDH1 enzyme function could not reverse the defect in reductive carboxylation activity. Furthermore, this metabolic reprogramming increased the sensitivity of IDH1-mutant cells to hypoxia or electron transport chain inhibition in vitro. Lastly, IDH1-mutant cells also grew poorly as subcutaneous xenografts within a hypoxic in vivo microenvironment. Together, our results suggest therapeutic opportunities to exploit the metabolic vulnerabilities specific to IDH1 mutation.

Smac mimetics: implications for enhancement of targeted therapies in leukemia

Drug resistance is a growing concern with clinical use of tyrosine kinase inhibitors. Utilizing in vitro models of intrinsic drug resistance and stromal-mediated chemoresistance, as well as functional mouse models of progressive and residual disease, we attempted to develop a potential therapeutic approach designed to suppress leukemia recurrence following treatment with selective kinase inhibitors. The novel inhibitor of apoptosis (IAP), LCL161, was observed to potentiate the effects of tyrosine kinase inhibition against leukemic disease both in the absence and presence of a stromal protected environment. LCL161 enhanced the proapoptotic effects of nilotinib and PKC412, against leukemic disease in vitro and potentiated the activity of both kinase inhibitors against leukemic disease in vivo. In addition, LCL161 synergized in vivo with nilotinib to reduce leukemia burden significantly below the baseline level suppression exhibited by a moderate-to-high dose of nilotinib. Finally, LCL161 displayed antiproliferative effects against cells characterized by intrinsic resistance to tyrosine kinase inhibitors as a result of expression of point mutations in the protein targets of drug inhibition. These results support the idea of using IAP inhibitors in conjunction with targeted tyrosine kinase inhibition to override drug resistance and suppress or eradicate residual disease.

Development of Peptidomimetics Targeting IAPs

Inhibitor of apoptosis proteins (IAPs) such as XIAP subvert apoptosis by binding and inhibiting caspases. Because occupation of the XIAP BIR3 peptide binding pocket by Smac abolishes the XIAP–caspase 9 interaction, it is a proapoptotic event of great therapeutic interest. An assay for pocket binding was developed based on the displacement of Smac 7-mer from BIR3. Through the physical and biochemical analysis of a variety of peptides, we have determined the minimum sequence required for inhibition of the Smac–BIR3 interaction and detailed the dimensions and topology of the BIR3 peptide binding pocket. This work describes the structure–activity relationship (SAR) for peptide inhibitors of Smac-IAP binding.

Toward the Validation of Maternal Embryonic Leucine Zipper Kinase: Discovery, Optimization of Highly Potent and Selective Inhibitors, and Preliminary Biology Insight.

MELK kinase has been implicated in playing an important role in tumorigenesis. Our previous studies suggested that MELK is involved in the regulation of cell cycle and its genetic depletion leads to growth inhibition in a subset of high MELK-expressing basal-like breast cancer cell lines. Herein we describe the discovery and optimization of novel MELK inhibitors 8a and 8b that recapitulate the cellular effects observed by short hairpin ribonucleic acid (shRNA)-mediated MELK knockdown in cellular models. We also discovered a novel fluorine-induced hydrophobic collapse that locked the ligand in its bioactive conformation and led to a 20-fold gain in potency. These novel pharmacological inhibitors achieved high exposure in vivo and were well tolerated, which may allow further in vivo evaluation.

Abstract B27: Correlation between TNFα and LCL161 anti‐tumor activity in patient derived xenograft models of human cancer

LCL161, a small molecule antagonist of inhibitor of apoptosis proteins (IAPs), induces TNF ‐mediated apoptosis in a subset of tumor cell lines including the MDA‐MB‐231 breast cancer line. To investigate the in vivo activity of LCL161, MDA‐MB‐231 tumor bearing mice were treated with a once weekly oral dose. We observed anti‐tumor activity that was associated with pharmacodynamic responses including degradation of CIAP1 and induction of cleaved caspase 3. The loss of CIAP1, specifically the E3 ligase activity of this protein, has been shown to directly impact the stability of a number of client proteins including NIK (NF‐κB inducing kinase), Mad1 (MAX‐binding protein), and others. Gene expression analysis on LCL161 treated MDA‐MB‐231 tumors revealed a striking upregulation of NF‐κB regulated target genes. These data were further supported by the observation that TNFα, a direct target of NF‐κB, was induced in LCL161 treated MDAMB‐231 tumor lysates. To explore whether TNFα expression levels could be used to predict single agent efficacy in a more clinically relevant context, a series of human primary tumor xenografts were profiled for baseline TNFα mRNA levels and evaluated for response to LCL161 in vivo. We observed that tumors with the highest TNFα expression levels showed the greatest sensitivity to LCL161. These studies demonstrate an essential role of the NF‐κB pathway in IAP antagonist anti‐tumor activity. Ongoing efforts focus on delineating the contribution of the canonical and non‐canonical NF‐κB pathways to efficacy and on leveraging this mechanism for LCL161 patient selection. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B27.

Abstract 138: Therapeutic targeting of inhibitor of apoptosis proteins

Inhibitor of Apoptosis Proteins (IAP) proteins negatively regulate cell death through a variety of mechanisms. The prototypical IAP family member XIAP binds and inhibits the catalytic activity of caspases 3/7 and caspase 9 via the BIR2-linker region and BIR3 domains, respectively. CIAP1 and CIAP2 do not directly inhibit caspases but negatively regulate death receptor mediated apoptosis via intrinsic E3 ligase activity towards RIPK and NIK among other client proteins. IAP inhibitors (IAPi) are low molecular weight compounds that mimic Smac and bind to the IAP binding motif in the BIR3 domain of XIAP, CIAP1 and CIAP2. Smac mimetics induce apoptosis as a single agent in a subset of tumor cell lines in vitro. Cell death is preceded by the rapid proteosome-mediated degradation of CIAP1 followed by activation of both canonical and non-canonical NFKB pathway activation, TNF production and robust activation of caspase 3/7 activity. Multiple nodes in the NFKB signaling pathway were interrogated following IAPi treatment in sensitive and resistant cancer cells to delineate the basis for differential responses. Although canonical and non-canonical NFKB signaling was activated in both sensitive and resistant cells, TNF was induced only in the former. LCL161 is a second generation orally bioavailable IAPi with nM affinity for XIAP, CIAP1, CIAP2. Consistent with above, tumor cell lines with high baseline TNF levels are predisposed to IAPi sensitivity. Curiously, addition of exogenous TNF can sensitize many otherwise resistant tumor cell lines, but not normal cells, to LCL161. We undertook an unbiased study of the entire TNF super family to determine what other TNF-like cytokines could sensitize tumor cells to LCL161- induced cell death. In addition to TNF, several cytokines synergized with LCL161 and in each case RIPK appeared to play a central role. LCL161 showed potent single agent activity in the MDA-MB-231 tumor xenograft model. In vivo efficacy was accompanied by a series of tumor pharmacodynamic readouts including CIAP1 elimination, activation of an NFKB transcriptional program and caspase activation. In primary patient derived human tumor xenograft models of triple negative breast cancer and NSCLC, LCL161 had a range of responses from no effect to tumor stasis. Consistent with in vitro mechanistic studies, tumor models which were sensitive had high basal TNF levels. LCL161 lacked single agent activity in the A2058 melanoma model but significantly enhanced the anti-tumor activity of paclitaxel. The LCL161-Taxol combination triggered synergistic activation of caspases and near complete regressions in xenograft tumors. Clinical trials in man with LCL161 are ongoing in patients with solid tumors. A range of pharmacodynamic readouts have been observed which are consistent with preclinical observations. These findings show promise for IAP inhibitor therapy in humans. 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 138.

“Addition” and “Subtraction”: Selectivity Design for Type II Maternal Embryonic Leucine Zipper Kinase Inhibitors

While adding the structural features that are more favored by on-target activity is the more common strategy in selectivity optimization, the opposite strategy of subtracting the structural features that contribute more to off-target activity can also be very effective. Reported here is our successful effort of improving the kinase selectivity of type II maternal embryonic leucine zipper kinase inhibitors by applying these two complementary approaches together, which clearly demonstrates the powerful synergy between them.

Abstract LB-139: IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Mutations in the genes encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a variety of tumor types, resulting in production of the proposed oncometabolite, 2-hydroxyglutarate (2-HG). How mutant IDH alters central carbon metabolism, though, remains unclear. To address this question, we performed 13C metabolic flux analysis (MFA) on an isogenic cell panel containing heterozygous IDH1/2 mutations. We observe a dramatic and consistent decrease in the ability of IDH1, but not IDH2, mutant cell lines to utilize reductive glutamine metabolism via the carboxylation of α-ketoglutarate to isocitrate. Additionally we find that cells with IDH1 mutations exhibit increased oxidative tricarboxylic acid (TCA) metabolism. Similar metabolic trends were observed in vivo as well, and also in endogenous, non-engineered IDH1/2 mutant cell lines. Interestingly, IDH1-mutant specific inhibitors were unable to reverse the decrease in reductive metabolism, suggesting that this metabolic phenotype is independent of 2-HG. Furthermore, this metabolic reprogramming increases the sensitivity of IDH1 mutant cells to hypoxia or electron transport chain (ETC) inhibition in vitro . IDH1 mutant cells also grow poorly as subcutaneous xenografts within hypoxic in vivo microenvironments. These results suggest that exploiting metabolic defects specific to IDH1 mutant cells could be an interesting avenue to explore therapeutically. Citation Format: Alexandra R. Grassian, Seth Parker, Shawn Davidson, Ajit Divakaruni, Courtney Green, Xiamei Zhang, Kelly Slocum, Minying Pu, Fallon Lin, Chad Vickers, Carol Joud-Caldwell, Franklin Chung, Hong Yin, Erika Handly, Christopher Straub, Joseph D. Growney, Matt Vander Heiden, Anne Murphy, Raymond Pagliarini, Christian Metallo. IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism. [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 LB-139. doi:10.1158/1538-7445.AM2014-LB-139

Abstract B159: Heterozygous IDH1 mutations modify the citric acid (TCA) cycle metabolism and sensitize cells to inhibition of mitochondrial respiration/oxidative phosphorylation.

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a variety of tumor types. Although these mutations are loss-of-function for conversion of isocitrate to α-ketoglutarate, the mutant enzymes greatly increase the production of the proposed oncometabolite, 2-hydroxyglutarate (2-HG). However the full metabolic consequences of IDH1/2 mutation in their heterozygous cellular context have yet to be fully explored. To address this question, we utilized a panel of isogenic cell lines with wild-type IDH1/2 or clinically relevant IDH1/2 mutations and examined the metabolic consequences of IDH mutation using (13)C metabolic flux analysis (MFA). We observe a dramatic and consistent decrease in the ability of IDH1 mutant cell lines to utilize reductive glutamine metabolism via the carboxylation of α-ketoglutarate back to isocitrate. This was not seen either in IDH2 mutant cell lines or in wild-type cell lines treated with exogenous 2-HG. Consistent with these changes, the IDH1 mutant cell lines, but not IDH2 mutant or 2-HG treated cells, were deficient in the utilization of glutamine for de novo lipogenesis. Similar trends were observed in endogenous, non-engineered IDH1/2 mutant cell lines. The decrease in reductive carboxylation in the IDH1 mutant cell lines raises the hypothesis that these cells may be more reliant on mitochondrial metabolism. Indeed, IDH1 mutant cells were more sensitive to either treatment with an electron transport chain inhibitor or growth in hypoxia (which also inhibits mitochondrial metabolism). These results show heterozygous IDH1 mutation robustly impacts wild-type cellular metabolism in a different manner than IDH2 mutation. Furthermore, these results suggest that IDH1 and IDH2 mutant tumors may be differentially sensitive to inhibitors of specific metabolic pathways. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B159. Citation Format: Alexandra R. Grassian, Seth Parker, Shawn Davidson, Courtney Green, Fallon Lin, Carol Joud-Caldwell, Hong Yin, Franklin Chung, Christopher Straub, Matthew Vander Heiden, Raymond Pagliarini, Christian Metallo. Heterozygous IDH1 mutations modify the citric acid (TCA) cycle metabolism and sensitize cells to inhibition of mitochondrial respiration/oxidative phosphorylation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B159.