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XLSX file - 132K, Table S2: Effects of EPZ-6438 on Gene Expression in EZH2 Mutant Lymphoma Cells.

PDF file - 811K, Fig. S1. Effects of EPZ-6438 on Gene Promoter H3K27 Methylation, Recruitment of PRC2 Components to Gene Promoters, and Cell Cycle, in WSU-DLCL2 Cells. Fig. S2: Effects of EPZ-6438 on Gene Expression in EZH2 Mutant Lymphoma Cell Lines. Fig. S3: Pharmacokinetic Profiles of EPZ-6438 in Rats and Mice. Fig. S4: EPZ-6438 Compound Levels in Plasma and WSU-DLCL2 Xenograft Tumor Homogenates from Mice Dosed for 7 or 28 Days. Fig. S5: In vivo Effects of EPZ-6438 in Lymphoma Xenograft Models. Fig. S6: Body Weights of Mice during Xenograft Efficacy Studies.

PDF file - 25K, Table S1: LCC Values for EPZ-6438 for WSU-DLCL2 Human Lymphoma Cells Dosed Either Continuously or After Compound Washout.

PDF file - 1496K, Supplementary Methods; Supplementary Figure 1: Production and characterisation of tumor cells dependent on Bcr-Abl for survival; Supplementary Figure 2: HDACi with different isoform specificities induce apoptosis of Bcr-Abl(p210) expressing cells; Supplementary Figure 3: Knockdown of HDAC6 does not induce the acetylation of histones or affect sensitivity to HDAC inhibitors; Supplementary Table 1: HDACi selectively inhibit a range of different HDACs; Supplementary Table 2: Pharmacokinetic analysis of vorinostat and MRLB-223; Supplementary Table 3: FDG uptake was measured at different times in the same animals using region of interest software (ROI) on the PET.

Histone deacetylase inhibitors (HDACi) are anticancer agents that induce hyperacetylation of histones, resulting in chromatin remodeling and transcriptional changes. In addition, nonhistone proteins, such as the chaperone protein Hsp90, are functionally regulated through hyperacetylation mediated by HDACis. Histone acetylation is thought to be primarily regulated by HDACs 1, 2, and 3, whereas the acetylation of Hsp90 has been proposed to be specifically regulated through HDAC6. We compared the molecular and biologic effects induced by an HDACi with broad HDAC specificity (vorinostat) with agents that predominantly inhibited selected class I HDACs (MRLB-223 and romidepsin). MRLB-223, a potent inhibitor of HDACs 1 and 2, killed tumor cells using the same apoptotic pathways as the HDAC 1, 2, 3, 6, and 8 inhibitor vorinostat. However, vorinostat induced histone hyperacetylation and killed tumor cells more rapidly than MRLB-223 and had greater therapeutic efficacy in vivo. FDCP-1 cells dependent on the Hsp90 client protein Bcr-Abl for survival, were killed by all HDACis tested, concomitant with caspase-dependent degradation of Bcr-Abl. These studies provide evidence that inhibition of HDAC6 and degradation of Bcr-Abl following hyperacetylation of Hsp90 is likely not a major mechanism of action of HDACis as had been previously posited. Mol Cancer Ther; 12(12); 2709–21. ©2013 AACR.

Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in these cancers. We have previously reported the discovery of EPZ005678 and EPZ-6438, potent and selective S-adenosyl-methionine-competitive small molecule inhibitors of EZH2. Although both compounds are similar with respect to their mechanism of action and selectivity, EPZ-6438 possesses superior potency and drug-like properties, including good oral bioavailability in animals. Here, we characterize the activity of EPZ-6438 in preclinical models of NHL. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 days after stopping compound treatment in two EZH2-mutant xenograft models. These data confirm the dependency of EZH2-mutant NHL on EZH2 activity and portend the utility of EPZ-6438 as a potential treatment for these genetically defined cancers. Mol Cancer Ther; 13(4); 842–54. ©2014 AACR.

Supplementary Data from Vorinostat Inhibits Brain Metastatic Colonization in a Model of Triple-Negative Breast Cancer and Induces DNA Double-Strand Breaks

Purpose: As chemotherapy and molecular therapy improve the systemic survival of breast cancer patients, the incidence of brain metastases increases. Few therapeutic strategies exist for the treatment of brain metastases because the blood-brain barrier severely limits drug access. We report the pharmacokinetic, efficacy, and mechanism of action studies for the histone deactylase inhibitor vorinostat (suberoylanilide hydroxamic acid) in a preclinical model of brain metastasis of triple-negative breast cancer.Experimental Design: The 231-BR brain trophic subline of the MDA-MB-231 human breast cancer cell line was injected into immunocompromised mice for pharmacokinetic and metastasis studies. Pharmacodynamic studies compared histone acetylation, apoptosis, proliferation, and DNA damage in vitro and in vivo.Results: Following systemic administration, uptake of [14C]vorinostat was significant into normal rodent brain and accumulation was up to 3-fold higher in a proportion of metastases formed by 231-BR cells. Vorinostat prevented the development of 231-BR micrometastases by 28% (P = 0.017) and large metastases by 62% (P < 0.0001) compared with vehicle-treated mice when treatment was initiated on day 3 post-injection. The inhibitory activity of vorinostat as a single agent was linked to a novel function in vivo: induction of DNA double-strand breaks associated with the down-regulation of the DNA repair gene Rad52.Conclusions: We report the first preclinical data for the prevention of brain metastasis of triple-negative breast cancer. Vorinostat is brain permeable and can prevent the formation of brain metastases by 62%. Its mechanism of action involves the induction of DNA double-strand breaks, suggesting rational combinations with DNA active drugs or radiation. (Clin Cancer Res 2009;15(19):6148–57)

Supplementary Figure 4 from Constitutive Activation of Signal Transducers and Activators of Transcription Predicts Vorinostat Resistance in Cutaneous T-Cell Lymphoma

Supplementary Figure 8 from Constitutive Activation of Signal Transducers and Activators of Transcription Predicts Vorinostat Resistance in Cutaneous T-Cell Lymphoma

Vorinostat is a histone deacetylase inhibitor that induces differentiation, growth arrest, and/or apoptosis of malignant cells both in vitro and in vivo and has shown clinical responses in ∼30% of patients with advanced mycosis fungoides and Sézary syndrome cutaneous T-cell lymphoma (CTCL). The purpose of this study was to identify biomarkers predictive of vorinostat response in CTCL using preclinical model systems and to assess these biomarkers in clinical samples. The signal transducer and activator of transcription (STAT) signaling pathway was evaluated. The data indicate that persistent activation of STAT1, STAT3, and STAT5 correlate with resistance to vorinostat in lymphoma cell lines. Simultaneous treatment with a pan-Janus-activated kinase inhibitor resulted in synergistic antiproliferative effect and down-regulation of the expression of several antiapoptotic genes. Immunohistochemical analysis of STAT1 and phosphorylated tyrosine STAT3 (pSTAT3) in skin biopsies obtained from CTCL patients enrolled in the vorinostat phase IIb trial showed that nuclear accumulation of STAT1 and high levels of nuclear pSTAT3 in malignant T cells correlate with a lack of clinical response. These results suggest that deregulation of STAT activity plays a role in vorinostat resistance in CTCL, and strategies that block this pathway may improve vorinostat response. Furthermore, these findings may be of prognostic value in predicting the response of CTCL patients to vorinostat. [Cancer Res 2008;68(10):3785–94]

Supplementary Figures 1-5, Tables 1-2 from Inhibition of NOTCH Signaling by Gamma Secretase Inhibitor Engages the RB Pathway and Elicits Cell Cycle Exit in T-Cell Acute Lymphoblastic Leukemia Cells

Supplementary Figure 7 from Constitutive Activation of Signal Transducers and Activators of Transcription Predicts Vorinostat Resistance in Cutaneous T-Cell Lymphoma

Supplementary Figure 6 from Constitutive Activation of Signal Transducers and Activators of Transcription Predicts Vorinostat Resistance in Cutaneous T-Cell Lymphoma

Supplementary Figure 1 from Constitutive Activation of Signal Transducers and Activators of Transcription Predicts Vorinostat Resistance in Cutaneous T-Cell Lymphoma

Supplementary Figure 5 from Constitutive Activation of Signal Transducers and Activators of Transcription Predicts Vorinostat Resistance in Cutaneous T-Cell Lymphoma

Supplementary Figure 3 from Constitutive Activation of Signal Transducers and Activators of Transcription Predicts Vorinostat Resistance in Cutaneous T-Cell Lymphoma

Supplementary Figure 2 from Constitutive Activation of Signal Transducers and Activators of Transcription Predicts Vorinostat Resistance in Cutaneous T-Cell Lymphoma

NOTCH signaling is deregulated in the majority of T-cell acute lymphoblastic leukemias (T-ALL) as a result of activating mutations in NOTCH1. Gamma secretase inhibitors (GSI) block proteolytic activation of NOTCH receptors and may provide a targeted therapy for T-ALL. We have investigated the mechanisms of GSI sensitivity across a panel of T-ALL cell lines, yielding an approach for patient stratification based on pathway activity and also providing a rational combination strategy for enhanced response to GSI. Whereas the NOTCH1 mutation status does not serve as a predictor of GSI sensitivity, a gene expression signature of NOTCH pathway activity does correlate with response, and may be useful in the selection of patients more likely to respond to GSI. Furthermore, inhibition of the NOTCH pathway activity signature correlates with the induction of the cyclin-dependent kinase inhibitors CDKN2D (p19INK4d) and CDKN1B (p27Kip1), leading to derepression of RB and subsequent exit from the cell cycle. Consistent with this evidence of cell cycle exit, short-term exposure of GSI resulted in sustained molecular and phenotypic effects after withdrawal of the compound. Combination treatment with GSI and a small molecule inhibitor of CDK4 produced synergistic growth inhibition, providing evidence that GSI engagement of the CDK4/RB pathway is an important mechanism of GSI action and supports further investigation of this combination for improved efficacy in treating T-ALL. [Cancer Res 2009;69(7):3060–8]

Targeting cytosolic nucleic acid sensing pathways to activate the Type I interferon (IFN) response is an emerging therapeutic strategy being explored in oncology. The PARP family consists of seventeen enzymes that regulate fundamental biological processes including response to cellular stress. PARP7 (TIPARP) is a stress-induced mono-ART that catalyzes the transfer of a single unit of ADP-ribose onto substrates (MARylation) to regulate their function and plays a role in suppressing the Type I IFN response in tumor cells (Gozgit 2021 Cancer Cell). RBN-2397 is the first potent and selective small molecule inhibitor of PARP7 catalytic function. To investigate the cell autonomous effects of PARP7 inhibition, we performed a cell line screen to identify PARP7 dependent cancer cell lines. We found that treatment of a subset of lines across several cancers led to a robust decrease in cell viability. Additionally, dosing of tumor bearing mice led to complete regressions in NCI-H1373 lung cancer xenografts. To investigate the mechanism of action (MOA) leading to decreased cell viability, we treated NCI-H1373 cells with RBN-2397 and found accumulation of cells in the G0/G1 phase of the cell cycle indicative of a cell cycle arrest. This arrest in NCI-H1373 cells was associated with the induction of senescence and increased mRNA expression of senescence associated secretory phenotype (SASP) genes. To evaluate the in vivo MOA, we performed an NCI-H1373 xenograft study and collected tumors after 7 days of RBN-2397 treatment. PARP7 inhibition led to decreased expression of Ki67, and increased expression of P21 and cleaved caspase-3, suggesting decreased proliferation and increased apoptosis. Increased expression of SASP genes was also observed in RBN-2397 treated tumors. Finally, we investigated transcriptional changes after RBN-2397 treatment by RNA sequencing. In addition to the effects observed in Type I IFN signaling, we also observed differential expression of genes associated with other pathways including autophagy and energy metabolism. Further evaluation of key autophagy proteins revealed that RBN-2397 affects autophagy flux and leads to a decrease in the oxygen consumption rate of cells and reduced ATP production from the mitochondria, suggesting that a change in energy metabolism may be related to the tumor intrinsic effect of RBN-2397. In summary, we show treatment of cancer cells with RBN-2397 not only leads to activation of tumor cell IFN signaling, but also causes G1 arrest and senescence, and changes in cancer cell autophagy and energy metabolism. In vivo, RBN-2397 treatment leads to complete tumor regressions in xenografts accompanied by decreased proliferation and increased apoptosis of tumor cells. RBN-2397 is currently being evaluated in the clinic as single agent in selected cancer types (NCT04053673) and in combination with anti-PD-1 therapies. Citation Format: Jennifer R. Molina, Joseph M. Gozgit, Melissa M. Vasbinder, Ryan P. Abo, Kaiko kunii, Kristy G. Kuplast-Barr, Bin Gui, Sunaina P. Nayak, Elena Minissale, Kerren K. Swinger, Tim J. Wigle, Alvin Z. Lu, Danielle J. Blackwell, Christina R. Majer, Yue Ren, Ellen Bamberg, Mario Niepel, Jan-Rung Mo, William D. Church, Ahmed S. Mady, Jeff Song, Zacharenia A. Varsamis, Luke Utley, Patricia E. Rao, Timoty J. Mitchison, Kevin W. Kuntz, Victoria M. Richon, Kristen McEachern, Heike Keilhack. PARP7 inhibitor RBN-2397 increases tumoral IFN signaling leading to various tumor cell intrinsic effects and tumor regressions in mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2154.

PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Here, we identify PARP7 as a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores type I interferon (IFN) signaling responses to nucleic acids in tumor models. Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression. Oral dosing of the PARP7 small-molecule inhibitor, RBN-2397, results in complete tumor regression in a lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model, dependent on inducing type I IFN signaling in tumor cells. PARP7 is a therapeutic target whose inhibition induces both cancer cell-autonomous and immune stimulatory effects via enhanced IFN signaling. These data support the targeting of a monoPARP in cancer and introduce a potent and selective PARP7 inhibitor to enter clinical development.

Summary PARP14 has been implicated by genetic knockout studies to promote protumor macrophage polarization and suppress the antitumor inflammatory response due to its role in modulating interleukin-4 (IL-4) and interferon-γ signaling pathways. Here, we describe structure-based design efforts leading to the discovery of a potent and highly selective PARP14 chemical probe. RBN012759 inhibits PARP14 with a biochemical half-maximal inhibitory concentration of 0.003 μM, exhibits >300-fold selectivity over all PARP family members, and its profile enables further study of PARP14 biology and disease association both in vitro and in vivo. Inhibition of PARP14 with RBN012759 reverses IL-4-driven protumor gene expression in macrophages and induces an inflammatory mRNA signature similar to that induced by immune checkpoint inhibitor therapy in primary human tumor explants. These data support an immune suppressive role of PARP14 in tumors and suggest potential utility of PARP14 inhibitors in the treatment of cancer.

Genomic instability in cancer cells leads to cellular stress through the accumulation of aberrant nucleic acid species in the cytosol. We have shown that PARP7, a monoPARP, is a negative regulator of cytosolic nucleic acid sensing in cancer cells. RBN-2397 is a potent and selective PARP7 inhibitor that induces antitumor immunity in preclinical models and is currently being evaluated in a Phase I clinical trial. In our preclinical investigations, we found that in a subset of cancer cell lines, such as NCI-H1373, inhibition of PARP7 triggers Type I IFN release, STAT1 phosphorylation, and growth arrest. In contrast, other cell lines, for example, HARA, do not mount an IFN-response upon PARP7 inhibition, even though they are responsive to transfection of exogenous nucleic acids and PARP7 is expressed and enzymatically active. To investigate the underlying mechanism of PARP7 inhibition and to determine the drivers of the differential sensitivity across cell lines we performed arrayed CRISPR knockout screens, targeting approximately 240 genes in the nucleic acid sensing and IFN signaling pathways, in the presence and absence of PARP7 inhibition. Our arrayed screens confirmed multiple hits from a previous genome-wide pooled synthetic/lethal CRISPR dropout screen. For example, targeting genes in the cGAS/STING pathway conferred resistance to PARP7 inhibition in the NCI-H1373 responder cells, suggesting a critical dependence on this sensing pathway. In the PARP7 inhibitor-resistant HARA cells, deletion of components of innate immune-signaling (such as AIM2 and ADAR1), the NF-κB pathway, and genes involved in autophagy sensitized the cells to PARP7 inhibition. We further delineated the function of PARP7 by comparing the effects of the CRISPR perturbation across different cellular readouts such as STAT1 phosphorylation, IFN release, and proliferation. With our work, we shed light on the mechanism by which PARP7 acts as a critical suppressor of the innate immune response. Our findings demonstrate both redundancy and crosstalk between different nucleic acid-sensing pathways and may explain why some cell lines are resistant to PARP7 inhibition. Citation Format: Bin Gui, Ryan Abo, Patrick Flynn, Alvin Z. Lu, Jan-Rung Mo, Joseph M. Gozgit, Melissa M. Vasbinder, Zacharenia A. Varsamis, Andrew Santospago, Victoria M. Richon, Kevin W. Kuntz, Heike Keilhack, Timothy J. Mitchison, Mario Niepel. Investigating the mechanism of PARP7 inhibition in type I interferon signaling by arrayed CRISPR screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1021.

Targeting cytosolic nucleic acid sensing pathways and the Type I interferon (IFN) response is an emerging therapeutic strategy being explored in oncology. The PARP family consists of seventeen enzymes that regulate fundamental biological processes including response to cellular stress. In contrast to PARP1, PARP7 (TIPARP) is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates (MARylation) to change their function and plays a role in suppressing the Type I IFN response. RBN-2397 selectively inhibits PARP7 compared to the approved PARP1 inhibitors and demonstrates > 50-fold selectivity for inhibition of PARP7 over all PARP family members as measured by biochemical assays. The inhibition of PARP1-mediated ADP-ribosylation has been well-characterized for several PARP1 inhibitors using a cellular hydrogen peroxide-induced PARylation assay. Here, we show that RBN-2397 inhibits PARP7-dependent MARylation with an IC50 of 2 nM exhibiting a 300-fold window over PARP1-driven PARylation. Using the mouse CT26 cell line, we showed that RBN-2397, but not the PARP1 inhibitor olaparib, induced Type I IFN signaling demonstrated by STAT1 phosphorylation. The effect on pSTAT1 was phenocopied by PARP7 knockout (KO). To further demonstrate specificity, we show that simultaneous KO of PARP7 prevented any additional increase of STAT1 phosphorylation by RBN-2397; however, KO of PARP1 had no effect on the induction of Type I IFN signaling by RBN-2397. We had previously reported that RBN-2397 dosing of CT26 tumor bearing immune competent BALB/c mice led to complete and durable tumor regressions which could be reversed by interfering with tumor-derived IFN signaling (1). Here we show that in contrast, RBN-2397 showed modest activity with no tumor regressions in CT26-tumor bearing immunodeficient NOG mice. To assess which immune cell populations are involved in the antitumor effects of RBN-2397, CT26 tumor-bearing BALB/c mice were depleted of CD4 T, CD8 T or NK cells. Depletion of CD4 T or NK cells had no effect on RBN-2397 antitumor activity; however, depletion of CD8 T cells significantly reversed the effects of RBN-2397, suggesting that CD8 T cells are responsible for much of the antitumor immunity induced by RBN-2397. We have discovered and developed RBN-2397, a first-in-class, potent and selective inhibitor of PARP7. We show RBN-2397 restores Type I IFN signaling in cancer cells and that this is an on-target effect of inhibiting the catalytic activity of PARP7 and not PARP1. We further show that the adaptive immune response was required for the antitumor effects of RBN-2397. RBN-2397 is the first agent to enter clinical trials that targets this tumor-intrinsic vulnerability, and a Phase I clinical trial is underway (NCT04053673). (1) AACR Jun 22-24, 2020: Cancer Res 2020;80 (16 Suppl): Abstract nr 3405. Citation Format: Joseph M. Gozgit, Melissa M. Vasbinder, Ryan P. Abo, Kaiko Kunii, Kristy G. Kuplast-Barr, Bin Gui, Alvin Z. Lu, Jennifer R. Molina, Elena Minissale, Kerren K. Swinger, Tim J. Wigle, Danielle J. Blackwell, Christina R. Majer, Yue Ren, Mario Niepel, Ellen Bamberg, Jan-Rung Mo, David Church, Jeff Song, Luke Utley, Patricia E. Rao, Timothy J. Mitchison, Kevin W. Kuntz, Victoria M. Richon, Heike Keilhack. RBN-2397: A potent and selective small molecule inhibitor of PARP7 that induces tumor-derived antitumor immunity dependent on CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 48.

CD38 is an ADP-ribosyl cyclase that converts NAD+ to ADP-ribose (ADPR) or cyclic ADPR (cADPR) and nicotinamide. The enzyme can exist in either an ecto- or endo-catalytic orientation with different sub-cellular localization, and therefore can regulate internal and external NAD+ pools. Both NAD+ and cADPR can impact T cell fitness and effector function, and CD38 has been shown to be increased in settings of chronic T cell activation. CD38 can mediate the non-canonical generation of the immune suppressive adenosine by catabolizing extracellular NAD+ resulting in immunosuppression in the microenvironment. Upon immune checkpoint inhibitor (ICI) therapy, CD38 is upregulated on cancer cells to drive ICI resistance. Therefore CD38, through its catalytic activity, has been implicated in tumor immune suppression and ICI resistance. Genetic knockout of CD38 has been shown to prevent tumor growth and improve T cell fitness. Here, we describe the effects of CD38 inhibition using a small molecule inhibitor on these key metabolites in various cellular and tumor models. RBN013209 is a potent and selective small molecule inhibitor of CD38 catalytic function. We demonstrate that inhibition of CD38 with RBN013209 prevents conversion of extracellular NAD+ to ADPR or cADPR in cancer cell lines and PBMCs. Similarly, RBN013209 inhibited intracellular CD38 activity and elevated intracellular NAD+ levels in cultured human primary T cells. Oral administration of RBN013209 to naïve mice resulted in dose-dependent elevation of NAD+ and reduction of ADPR in various tissues such as spleen and liver. We next assessed the expression of CD38 protein by immunohistochemistry following ICI treatment in various syngeneic cancer models to select a model for efficacy studies. We observed increases in CD38 expression on tumor cells and infiltrating immune cells in MC38 colon cancer and B16-F10 and Cloudman S91 melanoma models. In the MC38 tumor model, we observed single agent antitumor activity with RBN013209 that was associated with changes in NAD+ and ADPR. In B16-F10 tumor-bearing mice, we observed antitumor activity with RBN013209 in combination with anti-PD-L1 therapy. To evaluate CD38 as a biomarker in clinical samples, we assessed and confirmed the tumor expression of CD38 protein from lung, prostate and kidney cancer patients. Here, we show that inhibition of CD38 with a small molecule affects both intra- and extra-cellular CD38 activity and modulates key metabolites playing an important role in immunomodulation. Further, our data indicate that CD38 is increased by ICI treatment and that inhibition of CD38 can lead to antitumor activity. Citation Format: Prashant Shambharkar, Danielle J. Blackwell, Melissa M. Vasbinder, Laurie B. Schenkel, Kaiko Kunii, Jenkins L. Lemera, Kristy G. Kuplast-Barr, Yue Ren, Ellen Bamberg, W. David Church, Christina R. Majer, Luke Utley, Kristen McEachern, Mario Niepel, Tim J. Wigle, Kevin W. Kuntz, Victoria M. Richon, Heike Keilhack, Joseph M. Gozgit. Small molecule inhibitor of CD38 modulates its intra- and extracellular functions leading to antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1344.

PARPs (poly-ADP-ribose polymerases) are a family of enzymes that regulate a wide variety of important cellular processes including cellular stress signaling pathways implicated in inflammation and cancer. PARP14 is a member of the monoPARP sub-family of PARPs, which catalyze transfer of a single ADP ribose unit (mono-ADP-ribosylation or MARylation) to their substrates, unlike their polyPARP counterparts which construct chains of ADP ribose units (poly-ADP-ribosylation or PARylation). PARP14 is an interferon-stimulated gene (ISG) that is overexpressed in tumors compared to normal tissues and has been implicated by genetic knockout studies to promote pro-tumor macrophage polarization and suppress antitumor inflammatory response due to its role in modulating IL-4 and IFN-γ signaling pathways. Efforts to further explore and validate the role of PARP14 catalytic activity in these pathways have been hampered by a lack of potent, highly selective PARP14 inhibitors. Here we describe the discovery of the first such chemical probe, RBN012759, which inhibits PARP14 with an IC50 of 0.003 µM and exhibits >300-fold selectivity over all mono- and polyPARP family members. Medicinal chemistry efforts that began with an unselective micromolar screening hit were enabled by a detailed understanding of the PARP14 and broader PARP family binding pockets. X-ray co-crystal structures of RBN012759 and key early analogs bound to PARP14 clearly illustrate the origins of their potency and selectivity. RBN012759 is a cell permeable, soluble probe that demonstrates robust, dose-dependent stabilization of endogenous PARP14 and inhibition of MARylation in primary human macrophages. RBN012759 reverses IL-4 driven (pro-tumor) gene expression in macrophages, confirming published data generated with PARP14 KO and supporting an immune suppressive role of PARP14 in tumors. Moreover, we demonstrate that PARP14 inhibition in primary human tumor explants can induce an inflammatory mRNA signature similar to immune checkpoint therapy. Summary: Structure-based design of the first potent and highly selective inhibitor of PARP14, RBN012759, is described. The discovery of this chemical probe enabled exploration of the role of PARP14 in macrophage polarization and inflammatory pathways. RBN012759 reverses IL-4 driven gene expression in macrophages and induces an inflammatory mRNA signature in human tumor explants, data which support an immune suppressive role of PARP14 in tumors. Citation Format: Laurie Schenkel, Jennifer Molina, Kerren Swinger, Ryan Abo, Danielle Blackwell, Anne Cheung, William Church, Kristy Kuplast-Barr, Alvin Lu, Elena Minissale, Mario Niepel, Melissa Vasbinder, Tim Wigle, Victoria Richon, Heike Keilhack, Kevin Kuntz. A potent and selective PARP14 inhibitor decreases pro-tumor macrophage function and elicits inflammatory responses in tumor explants [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1038.

Targeting cytosolic nucleic acid sensing pathways and the Type I interferon (IFN) response is an emerging therapeutic strategy being explored in oncology. PARP7 is a member of the monoPARP class of enzymes, which catalyze the transfer of single units of ADP-ribose onto substrates to change their function. PARP7 expression is increased by cellular stress and aromatic hydrocarbons, and the PARP7 gene is amplified in cancers, especially in those of the upper aerodigestive tract. PARP7 has also been reported to negatively regulate the Type I IFN response by interacting with TBK1 during viral infection. Herein, we identify PARP7 as a novel negative regulator of cytosolic nucleic acid sensing in tumor cells. RBN-2397, is a potent and selective small molecule inhibitor of PARP7 catalytic function. We identified a subset of cancers exhibiting dependency on PARP7 for proliferation and found that cell lines with higher baseline expression of interferon stimulated genes were more sensitive. We further show that inhibition of PARP7 by RBN-2397 restores Type I IFN signaling as demonstrated by the induction of STAT1 phosphorylation and up-regulation of genes enriched for Type I IFN signaling in NCI-H1373 lung cancer cells. We examined the antitumor effects of once daily orally administered RBN-2397 in SCID mice with subcutaneous NCIH1373 xenograft tumors and observed a dose-dependent effect of RBN-2397 on tumor growth, with regressions at dose levels ≥30 mg/kg. To evaluate the antitumor immune response in vivo, we administered RBN-2397 to CT26 tumor-bearing, immunocompetent BALB/c mice, and observed significant tumor growth inhibition at all dose levels with complete and durable regressions in a subset of mice. All of these tumor-free mice rejected a challenge of injected CT26 cells, but were able to develop 4T1 tumors, demonstrating induction of tumor-specific adaptive immune memory. The antitumor effects of RBN-2397 were further enhanced when combined with an immune checkpoint inhibitor, anti-PD1. Using CRISPR-Cas9 to knockout either TBK1 or IFNAR1 in CT26 cells, we demonstrated that RBN-2397 antitumor immunity is dependent on the effects of tumor-derived Type I interferon on immune cells. Here, we show for the first time that cancer cells use PARP7 to suppress the Type I IFN response to cytosolic nucleic acids. We have discovered and developed RBN-2397, a first-in-class, potent and selective inhibitor of PARP7. We show RBN-2397 restores Type I IFN signaling in the tumor, causes complete tumor regressions and adaptive immunity in murine models. RBN-2397 is the first agent to enter clinical trials that targets this tumor-intrinsic vulnerability. Citation Format: Joseph M. Gozgit, Melissa M. Vasbinder, Ryan P. Abo, Kaiko Kunii, Kristy G. Kuplast-Barr, Bin Gui, Alvin Z. Lu, Kerren K. Swinger, Tim J. Wigle, Danielle J. Blackwell, Christina R. Majer, Yue Ren, Mario Niepel, Zacharenia A. Varsamis, Sunaina P. Nayak, Ellen Bamberg, Jan-Rung Mo, William Church, Jeff Song, Luke Utley, Patricia E. Rao, Timothy J. Mitchison, Kevin W. Kuntz, Victoria M. Richon, Heike Keilhack. PARP7 negatively regulates the type I interferon response in cancer cells and its inhibition leads to tumor regression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3405.

RBN-2397: A first-in-class PARP7 inhibitor targeting a newly discovered cancer vulnerability in stress-signaling pathways PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function (MARylation). PARP7 expression is increased by cellular stresses, including aromatic hydrocarbons and the PARP7 gene is amplified in cancers, especially in those of the upper aerodigestive tract. PARP7 has also been reported to negatively regulate the Type I interferon (IFN) response by interacting with TBK1 during viral infection. As part of our drug discovery efforts to identify inhibitors of PARP7, we utilized structure-based drug design to optimize an unselective monoPARP inhibitor identified by screening Ribon's internal compound collection of PARP inhibitors. Further optimization of potency and physicochemical properties led to the discovery of RBN-2397, a potent and selective small molecule inhibitor of PARP7 catalytic function. A co-crystal structure of RBN-2397 demonstrated binding of the compound in the NAD+-binding pocket. Binding to cellular PARP7 is demonstrated by the ability of RBN-2397 to displace an active site probe in a NanoBRET assay. Functionally, RBN-2397 leads to the inhibition of MARylation of multiple intracellular proteins in PARP7-overexpressing SK-MES-1 cells. We identified a subset of cancers exhibiting dependency on PARP7 for proliferation. Cell lines with higher baseline expression of interferon stimulated genes are more sensitive to RBN-2397 in proliferation assays. We further show that inhibition of PARP7 by RBN-2397 restores Type I IFN signaling as demonstrated by the induction of STAT1 phosphorylation and upregulation of genes enriched for Type I IFN signaling in NCI-H1373 lung cancer cells. Oral dosing of RBN-2397 results in durable, complete tumor regression in a NCI-H1373 lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model that is dependent on tumor-derived Type I IFN signaling. Herein, we describe the discovery of the small molecule PARP7 inhibitor RBN-2397, the first therapeutic agent targeting PARP7 to enter clinical trials, and the first disclosure of the inhibitor. We demonstrate PARP7 is a novel therapeutic target and inhibition of PARP7 by RBN-2397 induces both cancer cell autonomous and immune stimulatory effects via enhanced IFN signaling. Citation Format: Melissa M. Vasbinder, Joseph M. Gozgit, Ryan P. Abo, Kaiko Kunii, Kristy G. Kuplast-Barr, Bin Gui, Alvin Z. Lu, Kerren K. Swinger, Tim J. Wigle, Danielle J. Blackwell, Christina R. Majer, Yue Ren, Mario Niepel, Zacharenia A. Varsamis, Sunaina P. Nayak, Ellen Bamberg, Jan-Rung Mo, W David Church, Jeff Song, Luke Utley, Patricia E. Rao, Timothy J. Mitchison, Kevin W. Kuntz, Victoria M. Richon, Heike Keilhack. RBN-2397: A first-in-class PARP7 inhibitor targeting a newly discovered cancer vulnerability in stress-signaling pathways [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr DDT02-01.

Posttranslational methylation of histones plays a critical role in gene regulation. Misregulation of histone methylation can lead to oncogenic transformation. Enhancer of Zeste homologue 2 (EZH2) methylates histone 3 at lysine 27 (H3K27) and abnormal methylation of this site is found in many cancers. Tazemetostat, an EHZ2 inhibitor in clinical development, has shown activity in both preclinical models of cancer as well as in patients with lymphoma or INI1-deficient solid tumors. Herein we report the structure-activity relationships from identification of an initial hit in a high-throughput screen through selection of tazemetostat for clinical development. The importance of several methyl groups to the potency of the inhibitors is highlighted as well as the importance of balancing pharmacokinetic properties with potency.

The study by Kelly and colleagues, published in the September 1, 2003, issue of Clinical Cancer Research, established the safety and biologic activity of the first-in-class histone deacetylase inhibitor, vorinostat, which was administered intravenously. Subsequent studies led to the development of oral vorinostat and the regulatory approval of vorinostat for cutaneous T-cell lymphomas, which opened the door for the next generation of inhibitors. Clin Cancer Res; 21(10); 2198–200. ©2015 AACR. See related article by Kelly et al., Clin Cancer Res 2003;9(10) September 1, 2003;3578–88

(2R,3R,4S,5R)-2-(6-Amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol (EPZ-5676) is a novel DOT1L histone methyltransferase inhibitor currently in clinical development for the treatment of MLL-rearranged leukemias. This report describes the preclinical pharmacokinetics and metabolism of EPZ-5676, an aminonucleoside analog with exquisite target potency and selectivity that has shown robust and durable tumor growth inhibition in preclinical models. The in vivo pharmacokinetics in mouse, rat and dog were characterized following i.v. and p.o. administration; EPZ-5676 had moderate to high clearance, low oral bioavailability with a steady-state volume of distribution 2-3 fold higher than total body water. EPZ-5676 showed biexponential kinetics following i.v. administration, giving rise to a terminal elimination half-life (t1/2 ) of 1.1, 3.7 and 13.6 h in mouse, rat and dog, respectively. The corresponding in vitro ADME parameters were also studied and utilized for in vitro-in vivo extrapolation purposes. There was good agreement between the microsomal clearance and the in vivo clearance implicating hepatic oxidative metabolism as the predominant elimination route in preclinical species. Furthermore, low renal clearance was observed in mouse, approximating to fu -corrected glomerular filtration rate (GFR) and thus passive glomerular filtration. The metabolic pathways across species were studied in liver microsomes in which EPZ-5676 was metabolized to three monohydroxylated metabolites (M1, M3 and M5), one N-dealkylated product (M4) as well as an N-oxide (M6).

Histone deacetylase inhibitors (HDACi) are anticancer agents that induce hyperacetylation of histones, resulting in chromatin remodeling and transcriptional changes. In addition, nonhistone proteins, such as the chaperone protein Hsp90, are functionally regulated through hyperacetylation mediated by HDACis. Histone acetylation is thought to be primarily regulated by HDACs 1, 2, and 3, whereas the acetylation of Hsp90 has been proposed to be specifically regulated through HDAC6. We compared the molecular and biologic effects induced by an HDACi with broad HDAC specificity (vorinostat) with agents that predominantly inhibited selected class I HDACs (MRLB-223 and romidepsin). MRLB-223, a potent inhibitor of HDACs 1 and 2, killed tumor cells using the same apoptotic pathways as the HDAC 1, 2, 3, 6, and 8 inhibitor vorinostat. However, vorinostat induced histone hyperacetylation and killed tumor cells more rapidly than MRLB-223 and had greater therapeutic efficacy in vivo. FDCP-1 cells dependent on the Hsp90 client protein Bcr-Abl for survival, were killed by all HDACis tested, concomitant with caspase-dependent degradation of Bcr-Abl. These studies provide evidence that inhibition of HDAC6 and degradation of Bcr-Abl following hyperacetylation of Hsp90 is likely not a major mechanism of action of HDACis as had been previously posited. Mol Cancer Ther; 12(12); 2709–21. ©2013 AACR.

Rearrangements of the MLL gene define a genetically distinct subset of acute leukemias with poor prognosis. Current treatment options are of limited effectiveness; thus, there is a pressing need for new therapies for this disease. Genetic and small molecule inhibitor studies have demonstrated that the histone methyltransferase DOT1L is required for the development and maintenance of MLL-rearranged leukemia in model systems. Here we describe the characterization of EPZ-5676, a potent and selective aminonucleoside inhibitor of DOT1L histone methyltransferase activity. The compound has an inhibition constant value of 80 pM, and demonstrates 37 000-fold selectivity over all other methyltransferases tested. In cellular studies, EPZ-5676 inhibited H3K79 methylation and MLL-fusion target gene expression and demonstrated potent cell killing that was selective for acute leukemia lines bearing MLL translocations. Continuous IV infusion of EPZ-5676 in a rat xenograft model of MLL-rearranged leukemia caused complete tumor regressions that were sustained well beyond the compound infusion period with no significant weight loss or signs of toxicity. EPZ-5676 is therefore a potential treatment of MLL-rearranged leukemia and is under clinical investigation.

Tumor cells display fundamental changes in metabolism and nutrient uptake in order to utilize additional nutrient sources to meet their enhanced bioenergetic requirements. Glutamine (Gln) is one such nutrient that is rapidly taken up by tumor cells to fulfill this increased metabolic demand. A vital step in the catabolism of glutamine is its conversion to glutamate by the mitochondrial enzyme glutaminase (GLS). This study has identified GLS a potential therapeutic target in breast cancer, specifically in the basal subtype that exhibits a deregulated glutaminolysis pathway. Using inducible shRNA mediated gene knockdown, we discovered that loss of GLS function in triple-negative breast cancer (TNBC) cell lines with a deregulated glutaminolysis pathway led to profound tumor growth inhibition in vitro and in vivo. GLS knockdown had no effect on growth and metabolite levels in non-TNBC cell lines. We rescued the anti-tumor effect of GLS knockdown using shRNA resistant cDNAs encoding both GLS isoforms and by addition of an α-ketoglutarate (αKG) analog thus confirming the critical role of GLS in TNBC. Pharmacological inhibition of GLS with the small molecule inhibitor CB-839 reduced cell growth and led to a decrease in mammalian target of rapamycin (mTOR) activity and an increase in the stress response pathway driven by activating transcription factor 4 (ATF4). Finally, we found that GLS inhibition synergizes with mTOR inhibition, which introduces the possibility of a novel therapeutic strategy for TNBC. Our study revealed that GLS is essential for the survival of TNBC with a deregulated glutaminolysis pathway. The synergistic activity of GLS and mTOR inhibitors in TNBC cell lines suggests therapeutic potential of this combination for the treatment of vulnerable subpopulations of TNBC.

The human protein methyltransferases (PMTs) constitute a large enzyme class composed of two families, the protein lysine methyltransferases (PKMTs) and the protein arginine methyltransferases (PRMTs). Examples have been reported of both PKMTs and PRMTs that are genetically altered in specific human cancers, and in several cases these alterations have been demonstrated to confer a unique dependence of the cancer cells on PMT enzymatic activity for the tumorigenic phenotype. Examples of such driver alterations in PMTs will be presented together with a review of current efforts towards the discovery and development of small-molecule inhibitors of these enzymes as personalized cancer therapeutics.

EZH2 catalyzes trimethylation of histone H3 lysine 27 (H3K27). Point mutations of EZH2 at Tyr641 and Ala677 occur in subpopulations of non-Hodgkin's lymphoma, where they drive H3K27 hypertrimethylation. Here we report the discovery of EPZ005687, a potent inhibitor of EZH2 (K(i) of 24 nM). EPZ005687 has greater than 500-fold selectivity against 15 other protein methyltransferases and has 50-fold selectivity against the closely related enzyme EZH1. The compound reduces H3K27 methylation in various lymphoma cells; this translates into apoptotic cell killing in heterozygous Tyr641 or Ala677 mutant cells, with minimal effects on the proliferation of wild-type cells. These data suggest that genetic alteration of EZH2 (for example, mutations at Tyr641 or Ala677) results in a critical dependency on enzymatic activity for proliferation (that is, the equivalent of oncogene addiction), thus portending the clinical use of EZH2 inhibitors for cancers in which EZH2 is genetically altered.

A variety of molecular mechanisms work together to effect strict control of gene transcription in human cells. Paramount among these mechanisms is a collection of post-translational modifications of chromatin that facilitate conformational transitions between transcriptionally permissive and suppressive chromatin structures (Figure 1; refs. 1, 2). Each of these modifications is catalyzed by a specific set of enzymes (Figure 2). Not surprisingly, dysregulation of chromatin modification leads to pathologic alterations in gene transcription, hence to human disease. Among chromatin modifying enzymes, the protein methyltransferases (PMTs) stand out due to the large size of the class, and the disease association of many of these enzymes (3).

SummaryThe histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been implicated in the development of leukemias bearing translocations of the Mixed Lineage Leukemia (MLL) gene. We identified the MLL-fusion targets in an MLL-AF9 leukemia model, and conducted epigenetic profiling for H3K79me2, H3K4me3, H3K27me3, and H3K36me3 in hematopoietic progenitor and leukemia stem cells (LSCs). We found abnormal profiles only for H3K79me2 on MLL-AF9 fusion target loci in LSCs. Inactivation of Dot1l led to downregulation of direct MLL-AF9 targets and an MLL translocation-associated gene expression signature, whereas global gene expression remained largely unaffected. Suppression of MLL translocation-associated gene expression corresponded with dependence of MLL-AF9 leukemia on Dot1l in vivo. These data point to DOT1L as a potential therapeutic target in MLL-rearranged leukemia.

A survey of the human genome was performed to understand the constituency of protein methyltransferases (both protein arginine and lysine methyltransferases) and the relatedness of their catalytic domains. We identified 51 protein lysine methyltransferase proteins based on similarity to the canonical Drosophila Su(var)3-9, enhancer of zeste (E(z)), and trithorax (trx) domain. Disruptor of telomeric silencing-1-like, a known protein lysine methyltransferase, did not fit within the protein lysine methyltransferase family, but did group with the protein arginine methyltransferases, along with 44 other proteins, including the METTL and NOP2/Sun domain family proteins. We show that a representative METTL, METTL11A, demonstrates catalytic activity as a histone methyltransferase. We also solved the co-crystal structures of disruptor of telomeric silencing-1-like with S-adenosylmethionine and S-adenosylhomocysteine bound in its active site. The conformation of both ligands is virtually identical to that found in known protein arginine methyltransferases, METTL and NOP2/Sun domain family proteins and is distinct from that seen in the Drosophila Su(var)3-9, enhancer of zeste (E(z)), and trithorax (trx) domain protein lysine methyltransferases. We have developed biochemical assays for 11 members of the protein methyltransferase target class and have profiled the affinity of three ligands for these enzymes: the common methyl-donating substrate S-adenosylmethionine; the common reaction product S-adenosylhomocysteine; and the natural product sinefungin. The affinity of each of these ligands is mapped onto the family trees of the protein lysine methyltransferases and protein arginine methyltransferases to reveal patterns of ligand recognition by these enzymes.

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1–5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.

EZH2, the catalytic subunit of the PRC2 complex, catalyzes the mono- through trimethylation of lysine 27 on histone H3 (H3K27). Histone H3K27 trimethylation is a mechanism for suppressing transcription of specific genes that are proximal to the site of histone modification. Point mutations of the EZH2 gene (Tyr641) have been reported to be linked to subsets of human B-cell lymphoma. The mutant allele is always found associated with a wild-type allele (heterozygous) in disease cells, and the mutations were reported to ablate the enzymatic activity of the PRC2 complex for methylating an unmodified peptide substrate. Here we demonstrate that the WT enzyme displays greatest catalytic efficiency ( k cat / K ) for the zero to monomethylation reaction of H3K27 and diminished efficiency for subsequent (mono- to di- and di- to trimethylation) reactions. In stark contrast, the disease-associated Y641 mutations display very limited ability to perform the first methylation reaction, but have enhanced catalytic efficiency for the subsequent reactions, relative to the WT enzyme. These results imply that the malignant phenotype of disease requires the combined activities of a H3K27 monomethylating enzyme (PRC2 containing WT EZH2 or EZH1) together with the mutant PRC2s for augmented conversion of H3K27 to the trimethylated form. To our knowledge, this is the first example of a human disease that is dependent on the coordinated activities of normal and disease-associated mutant enzymatic function.

Tumor cells display enhanced requirements for energy and building blocks such as amino acids, nucleic acids and lipids to sustain their proliferation. Metabolic demands of cancer cells are often met in the context of limited access to nutrients and the need to maintain redox balance. Glutamine catabolism is considered critical to cancer cell survival by not only delivering carbon and nitrogen atoms needed for nucleic acid and amino acid precursors but also by serving as the source of antioxidants such as NADPH and glutathione. A vital step in the catabolism of glutamine is its conversion to glutamate by the mitochondrial enzyme glutaminase (GLS).In this study, we confirmed the significance of GLS and enhanced glutamine utilization in the basal subtype of breast cancer as evidenced by higher GLS to GLUL (Glutamine synthetase) ratio in a panel of breast cancers in The Cancer Genome Atlas (TCGA) database. Using inducible shRNA mediated gene knockdown, we discovered that loss of GLS function in triple-negative breast cancer (TNBC) cell lines led to profound tumor growth inhibition in vitro and in vivo. This anti-tumor effect of GLS knockdown was rescued either using shRNA resistant cDNAs encoding both GLS isoforms or by addition of an a-ketoglutarate analog thus confirming the critical role of GLS in TNBC. Along with these observations, we have found that pharmacological inhibition of GLS with the small molecule inhibitor CB-839 reduced cell growth and led to a decrease in mammalian target of rapamycin (mTOR) coupled with an increase in the stress response pathway driven by activating transcription factor 4 (ATF4). In line with the observed impact on mTOR pathway we found that GLS inhibition synergizes with mTOR inhibition. In conclusion, our preliminary investigation revealed that glutaminolysis is deregulated in a majority of TNBC cancer patients. Our target credentialing studies confirmed that GLS is essential for the survival of TNBC cell lines and xenografts tumors. The synergistic activity of GLS and mTOR inhibitors in this specific breast cancer subtype suggests a new therapeutic modality that may bring benefit to TNBC patients with high unmet need. Citation Format: Christophe Henry, Michael Lampa, Timothy He, Beatriz Ospina, Bailin Zhang, Gejing Deng, Claude Barberis, Dietmar Hoffmann, Jack Pollard, Adrian Francisco, Heike Arlt, Jason Reeves, Joshua Murtie, Christopher Winter, Victoria Richon, Hong Cheng, Carlos Garcia-Echeverria, Dmitri Wiederschain, Lakshmi Srinivasan. Basal-like breast cancer subtype is characterized by deregulated glutamine metabolism and is sensitive to GLS inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5472.

Vorinostat (SAHA, Zolinza), a histone deacetylase inhibitor, is assessed in nonclinical studies to support its approval for cutaneous T-cell lymphoma. Vorinostat is weakly mutagenic in the Ames assay; is clastogenic in rodent (ie, CHO) cells but not in normal human lymphocytes; and is weakly positive in an in vivo mouse micronucleus assay. No effects are observed on potassium ion currents in the hERG assay up to 300 microM (safety margin approximately 300-fold the approximately 1 microM serum concentration associated with the 400 mg/d maximum recommended human dose. No rat respiratory or central nervous system effects are found at 150 mg/kg (2-fold maximum recommended human dose). No cardiovascular effects, including effects on QTc interval, are observed after a single oral dose (150 mg/kg) in dogs. Vorinostat is orally dosed daily in rats (controls, 20, 50, or 150 mg/kg/d) and dogs (controls, 60, 80, or 100/125/160 mg/kg/d) for 26 weeks with a 4-week recovery. Rat vorinostat-related adverse findings are decreased food consumption, weight loss, and hematologic changes; a no observed adverse effects level is not established. In dogs, adverse effects are primarily gastrointestinal; the no observed adverse effects level is 60 mg/kg/d (approximately 6-fold maximum recommended human dose). Toxicities are reversible and can be monitored in the clinic.

Purpose: As chemotherapy and molecular therapy improve the systemic survival of breast cancer patients, the incidence of brain metastases increases. Few therapeutic strategies exist for the treatment of brain metastases because the blood-brain barrier severely limits drug access. We report the pharmacokinetic, efficacy, and mechanism of action studies for the histone deactylase inhibitor vorinostat (suberoylanilide hydroxamic acid) in a preclinical model of brain metastasis of triple-negative breast cancer. Experimental Design: The 231-BR brain trophic subline of the MDA-MB-231 human breast cancer cell line was injected into immunocompromised mice for pharmacokinetic and metastasis studies. Pharmacodynamic studies compared histone acetylation, apoptosis, proliferation, and DNA damage in vitro and in vivo. Results: Following systemic administration, uptake of [14C]vorinostat was significant into normal rodent brain and accumulation was up to 3-fold higher in a proportion of metastases formed by 231-BR cells. Vorinostat prevented the development of 231-BR micrometastases by 28% (P = 0.017) and large metastases by 62% (P < 0.0001) compared with vehicle-treated mice when treatment was initiated on day 3 post-injection. The inhibitory activity of vorinostat as a single agent was linked to a novel function in vivo: induction of DNA double-strand breaks associated with the down-regulation of the DNA repair gene Rad52. Conclusions: We report the first preclinical data for the prevention of brain metastasis of triple-negative breast cancer. Vorinostat is brain permeable and can prevent the formation of brain metastases by 62%. Its mechanism of action involves the induction of DNA double-strand breaks, suggesting rational combinations with DNA active drugs or radiation. (Clin Cancer Res 2009;15(19):6148–57)

Epigenetic abnormalities are common in human cancer and play a key role in tumor progression through dysregulation of gene expression and chromatin function. The recent identification of the enzymes regulating epigenetic modifications and their altered function in cancer supports the possibility that these abnormalities can be reversed by inhibiting these enzymes. Indeed, drugs targeting such enzymes including DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) have demonstrated antitumor activity in the clinic and inhibitors of DNMTs and HDACs are now approved as anticancer agents. As our understanding of cancer epigenetics grows, members of additional enzyme classes such as histone methyltransferases and demethylases are emerging as targets for future epigenetic cancer therapies.

Inhibitors of class 1 and class 2 histone deacetylase (HDAC) enzymes have shown antitumor activity in human clinical trials. More recently, there has been interest in developing subtype-selective HDAC inhibitors designed to retain anticancer activity while reducing potential side effects. Efforts have been initiated to selectively target HDAC1 given its role in tumor proliferation and survival. The development of HDAC1-specific inhibitors will require the identification of HDAC1-selective pharmacodynamic markers that correlate closely with HDAC1-inhibition in vitro and in vivo. Existing histone markers of HDAC target engagement were developed using pan-HDAC inhibitors and do not necessarily represent robust readouts for isoform-specific inhibitors. Therefore, we have initiated a proteomic approach to identify readouts for HDAC1 inhibition. This approach involves the use of differential mass spectrometry (dMS) to identify post-translational changes in histones by profiling histone-enriched cellular fractions treated with various HDAC inhibitors. In this study, we profiled histones isolated from the HCT116 human colon cancer cell line that have been treated with compounds from multiple chemical classes that are specific for HDAC1; HDAC1 and 3; and HDAC1, 3, and 6 enzymes. In two independent experiments, we identified 24 features that correlated with HDAC1-inhibition. Among the peptides modulated by HDAC1-selective inhibitors were Ac-H2B-K5 from histone H2B, and Ac-H3-K18 from histone H3. Commercially available antibodies to specific histone acetyl-lysine residues were used to confirm that these peptides also provide pharmacodynamic readouts for HDAC1-selective inhibitors in vivo and in vitro. These results show the utility of dMS in guiding the identification of specific readouts to aid in the development of HDAC-selective inhibitors.

Purpose: For many tumor cells, de novo lipogenesis is a requirement for growth and survival. A considerable body of work suggests that inhibition of this pathway may be a powerful approach to antineoplastic therapy. It has recently been shown that inhibition of various steps in the lipogenic pathway individually can induce apoptosis or loss of viability in tumor cells. However, it is not clear whether quantitative differences exist in the ability of lipogenic enzymes to control tumor cell survival. We present a systematic approach that allows for a direct comparison of the control of lipogenic pathway enzymes over tumor cell growth and apoptosis using different cancer cells. Experimental Design: RNA interference-mediated, graded down-regulation of fatty acid synthase (FAS) pathway enzymes was employed in combination with measurements of lipogenesis, apoptosis, and cell growth. Results: In applying RNA interference titrations to two lipogenic enzymes, acetyl-CoA carboxylase 1 (ACC1) and FAS, we show that ACC1 and FAS both significantly control cell growth and apoptosis in HCT-116 cells. These results also extend to PC-3 and A2780 cancer cells. Conclusions: Control of tumor cell survival by different steps in de novo lipogenesis can be quantified. Because ACC1 and FAS both significantly control tumor cell growth and apoptosis, we propose that pharmacologic inhibitors of either enzyme might be useful agents in targeting cancer cells that critically rely on fatty acid synthesis. The experimental approach described here may be extended to other targets or disease-relevant pathways to identify steps suitable for therapeutic intervention.

Vorinostat is a histone deacetylase inhibitor that induces differentiation, growth arrest, and/or apoptosis of malignant cells both in vitro and in vivo and has shown clinical responses in ∼30% of patients with advanced mycosis fungoides and Sézary syndrome cutaneous T-cell lymphoma (CTCL). The purpose of this study was to identify biomarkers predictive of vorinostat response in CTCL using preclinical model systems and to assess these biomarkers in clinical samples. The signal transducer and activator of transcription (STAT) signaling pathway was evaluated. The data indicate that persistent activation of STAT1, STAT3, and STAT5 correlate with resistance to vorinostat in lymphoma cell lines. Simultaneous treatment with a pan-Janus-activated kinase inhibitor resulted in synergistic antiproliferative effect and down-regulation of the expression of several antiapoptotic genes. Immunohistochemical analysis of STAT1 and phosphorylated tyrosine STAT3 (pSTAT3) in skin biopsies obtained from CTCL patients enrolled in the vorinostat phase IIb trial showed that nuclear accumulation of STAT1 and high levels of nuclear pSTAT3 in malignant T cells correlate with a lack of clinical response. These results suggest that deregulation of STAT activity plays a role in vorinostat resistance in CTCL, and strategies that block this pathway may improve vorinostat response. Furthermore, these findings may be of prognostic value in predicting the response of CTCL patients to vorinostat. [Cancer Res 2008;68(10):3785–94]

Aims: Vorinostat (suberoylanilide hydroxamic acid; SAHA) is a histone deacetylase inhibitor (HDACi) approved in the clinics for the treatment of T-cell lymphoma and with the potential to be effective also in breast cancer. We investigated the responsiveness to SAHA in human breast primary tumors and cancer cell lines. Results: We observed a differential response to drug treatment in both human breast primary tumors and cancer cell lines. Gene expression analysis of the breast cancer cell lines revealed that genes involved in cell adhesion and redox pathways, especially glutathione metabolism, were differentially expressed in the cell lines resistant to SAHA compared with the sensitive ones, indicating their possible association with drug resistance mechanisms. Notably, such an association was also observed in breast primary tumors. Indeed, addition of buthionine sulfoximine (BSO), a compound capable of depleting cellular glutathione, significantly enhanced the cytotoxicity of SAHA in both breast cancer cell lines and primary breast tumors. Innovation: We identify and validate transcriptional differences in genes involved in redox pathways, which include potential predictive markers of sensitivity to SAHA. Conclusion: In breast cancer, it could be relevant to evaluate the expression of antioxidant genes that may favor tumor resistance as a factor to consider for potential clinical application and treatment with epigenetic drugs (HDACis). Antioxid. Redox Signal. 23, 15–29.