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Supplementary Figure 3 from MK-2461, a Novel Multitargeted Kinase Inhibitor, Preferentially Inhibits the Activated c-Met Receptor

Supplementary Figures 1-4 from PDK1 Attenuation Fails to Prevent Tumor Formation in PTEN-Deficient Transgenic Mouse Models

Supplementary Figure 4 from MK-2461, a Novel Multitargeted Kinase Inhibitor, Preferentially Inhibits the Activated c-Met Receptor

Supplementary Figure 2 from MK-2461, a Novel Multitargeted Kinase Inhibitor, Preferentially Inhibits the Activated c-Met Receptor

Supplementary Figure 1 from MK-2461, a Novel Multitargeted Kinase Inhibitor, Preferentially Inhibits the Activated c-Met Receptor

Supplementary Figure 5 from MK-2461, a Novel Multitargeted Kinase Inhibitor, Preferentially Inhibits the Activated c-Met Receptor

We have identified a critical role for amplified FGFR2 in gastric cancer cell proliferation and survival. In a panel of gastric cancer cell lines, fibroblast growth factor receptor 2 (FGFR2) was overexpressed and tyrosine phosphorylated selectively in FGFR2-amplified cell lines KatoIII, Snu16, and OCUM-2M. FGFR2 kinase inhibition by a specific small-molecule inhibitor resulted in selective and potent growth inhibition in FGFR2-amplified cell lines, resulting in growth arrest in KatoIII cells and prominent induction of apoptosis in both Snu16 and OCUM-2M cells. FGFR2-amplified cell lines also contained elevated phosphotyrosine in EGFR, Her2, and Erbb3, but the elevated phosphorylation in EGFR could not be inhibited by gefitinib or erlotinib. We show that the elevated EGFR, Her2, and Erbb3 phosphotyrosine is dependent on FGFR2, revealing EGFR family kinases to be downstream targets of amplified FGFR2. Moreover, shRNA to Erbb3 resulted in a loss of proliferation, confirming a functional role for the activated EGFR signaling pathway. These results reveal that both the FGFR2 and EGFR family signaling pathways are activated in FGFR2-amplified gastric cancer cell lines to drive cell proliferation and survival. Inhibitors of FGFR2 or Erbb3 signaling may have therapeutic efficacy in the subset of gastric cancers containing FGFR2 amplification. [Cancer Res 2008;68(7):2340–8]

Supplementary Figures 1-10 from FGFR2-Amplified Gastric Cancer Cell Lines Require FGFR2 and Erbb3 Signaling for Growth and Survival

Supplementary Methods, Table 1, Figure Legends 1-5 from MK-2461, a Novel Multitargeted Kinase Inhibitor, Preferentially Inhibits the Activated c-Met Receptor

Adaptive resistance limits immune checkpoint blockade therapy (ICBT) response duration and magnitude. Interferon γ (IFNγ), a critical cytokine that promotes cellular immunity, also induces adaptive resistance to ICBT. Using syngeneic mouse tumour models, we confirmed that chronic IFNγ exposure confers resistance to anti-Programmed cell death protein 1 (α-PD-1) therapy. We identified consistent upregulation of poly-ADP ribosyl polymerase 14 (PARP14) in both chronic IFNγ-treated cancer cells and patient melanoma with elevated IFNG expression. Knockdown or pharmacological inhibition of PARP14 increased effector T cell infiltration into tumours derived from cells pre-treated with IFNγ and decreased the presence of regulatory T cells, leading to restoration of α-PD-1 sensitivity. Finally, we determined that tumours which spontaneously relapsed following α-PD-1 therapy could be re-sensitised upon receiving PARP14 inhibitor treatment, establishing PARP14 as an actionable target to reverse IFNγ-driven ICBT resistance.

The type 2 cytokines IL-4 and IL-13, which share use of an IL-4 receptor alpha chain and its nuclear induction of the transcription factor STAT6, are crucial in elicitation and maintenance of allergic conditions that include asthma. Prior work has shown a physical and functional association of STAT6 with PARP14, an ADP-ribosyl monotransferase. Moreover, elimination of all PARP14 expression by gene targeting led to altered recall antibody responses and attenuation of ovalbumin-specific allergic lung inflammation with no apparent health issues for mice lacking this protein. However, an unanswered question is whether or not inhibition of the catalytic function has any biological consequence since PARP14 has multiple functional domains apart from the portion that catalyzes ADP-ribosylation. As reported separately, iterative structural analyses and medicinal chemistry fostered the generation of a compound, RBN2759, that is highly selective in its inhibition of PARP14 with negligible impact on other members of the PARP gene family. We show here that administration of this compound to mice previously sensitized to the allergenAlternaria alternataachieved biochemically active levels and altered physiological responses to the antigen. These results show for the first time that in vivo administration of a specific inhibitor of the ADP-ribosyltransferase activity encoded by PARP14 is sufficient to alter biological responses. Specifically, the orally absorbable pharmaceutical compound decreased allergen-induced mucus, blunted the induced increases in circulating IgE, and prevented suppression of IgG2a. We conclude that the catalytic activity can contribute to pathogenesis in allergic processes and propose that other biological endpoints that depend on ADP-ribosylation by PARP14 can be targeted using selective inhibition.

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.

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.

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.

IDHology Among the most exciting drug targets to emerge from cancer genome sequencing projects are two related metabolic enzymes, isocitrate dehydrogenases 1 and 2 (IDH1, IDH2). Mutations in the IDH1 and IDH2 genes are common in certain types of human cancer. Whether inhibition of mutant IDH activity might offer therapeutic benefits is unclear (see the Perspective by Kim and DeBerardinis ). F. Wang et al. (p. 622 , published online 4 April) isolated a small molecule that selectively inhibits mutant IDH2, describe the structural details of its binding to the mutant enzyme, and show that this compound suppresses the growth of patient-derived leukemia cells harboring the IDH2 mutation. Rohle et al. (p. 626 , published online 4 April) show that a small molecule inhibitor of IDH1 selectively slows the growth of patient-derived brain tumor cells with the IDH1 mutation.

Activation of the phosphoinositide 3-kinase pathway is commonly observed in human prostate cancer. Loss of function of phosphatase and tensin homolog (PTEN) is associated with the activation of AKT and mammalian target of rapamycin (mTOR) in many cancer cell lines as well as in other model systems. However, activation of mTOR is also dependent of kinases other than AKT. Here, we show that activation of mTOR is not dependent on AKT in a prostate-specific PTEN-deficient mouse model of prostate cancer. Pathway bifurcation of AKT and mTOR was noted in both mouse and human prostate tumors. We demonstrated for the first time that cotargeting mTOR and AKT with ridaforolimus/MK-8669 and M1K-2206, respectively, delivers additive antitumor effects in vivo when compared to single agents. Our preclinical data suggest that the combination of AKT and mTOR inhibitors might be more effective in treating prostate cancer patients than current treatment regimens or either treatment alone.

SummaryProliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM2 isoform, effectively constraining lower glycolysis. Here, we report the discovery of PKM2 activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM2 activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the nonessential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM2 activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM2 expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress.

c-Met is a transmembrane tyrosine kinase that mediates activation of several signaling pathways implicated in aggressive cancer phenotypes. In recent years, research into this area has highlighted c-Met as an attractive cancer drug target, triggering a number of approaches to disrupt aberrant c-Met signaling. Screening efforts identified a unique class of 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one kinase inhibitors, exemplified by 1. Subsequent SAR studies led to the development of 81 (MK-2461), a potent inhibitor of c-Met that was efficacious in preclinical animal models of tumor suppression. In addition, biochemical studies and X-ray analysis have revealed that this unique class of kinase inhibitors binds preferentially to the activated (phosphorylated) form of the kinase. This report details the development of 81 and provides a description of its unique biochemical properties.

Background and purpose: γ-Secretase inhibitors (GSIs) block NOTCH receptor cleavage and pathway activation and have been under clinical evaluation for the treatment of malignancies such as T-cell acute lymphoblastic leukaemia (T-ALL). The ability of GSIs to decrease T-ALL cell viability in vitro is a slow process requiring >8 days, however, such treatment durations are not well tolerated in vivo. Here we study GSI's effect on tumour and normal cellular processes to optimize dosing regimens for anti-tumour efficacy. Experimental approach: Inhibition of the Notch pathway in mouse intestinal epithelium was used to evaluate the effect of GSIs and guide the design of dosing regimens for xenograft models. Serum Aβ40 and Notch target gene modulation in tumours were used to evaluate the degree and duration of target inhibition. Pharmacokinetic and pharmacodynamic correlations with biochemical, immunohistochemical and profiling data were used to demonstrate GSI mechanism of action in xenograft tumours. Key results: Three days of >70% Notch pathway inhibition was sufficient to provide an anti-tumour effect and was well tolerated. GSI-induced conversion of mouse epithelial cells to a secretory lineage was time- and dose-dependent. Anti-tumour efficacy was associated with cell cycle arrest and apoptosis that was in part due to Notch-dependent regulation of mitochondrial homeostasis. Conclusions and implications: Intermittent but potent inhibition of Notch signalling is sufficient for anti-tumour efficacy in these T-ALL models. These findings provide support for the use of GSI in Notch-dependent malignancies and that clinical benefits may be derived from transient but potent inhibition of Notch.

We have identified a critical role for amplified FGFR2 in gastric cancer cell proliferation and survival. In a panel of gastric cancer cell lines, fibroblast growth factor receptor 2 (FGFR2) was overexpressed and tyrosine phosphorylated selectively in FGFR2-amplified cell lines KatoIII, Snu16, and OCUM-2M. FGFR2 kinase inhibition by a specific small-molecule inhibitor resulted in selective and potent growth inhibition in FGFR2-amplified cell lines, resulting in growth arrest in KatoIII cells and prominent induction of apoptosis in both Snu16 and OCUM-2M cells. FGFR2-amplified cell lines also contained elevated phosphotyrosine in EGFR, Her2, and Erbb3, but the elevated phosphorylation in EGFR could not be inhibited by gefitinib or erlotinib. We show that the elevated EGFR, Her2, and Erbb3 phosphotyrosine is dependent on FGFR2, revealing EGFR family kinases to be downstream targets of amplified FGFR2. Moreover, shRNA to Erbb3 resulted in a loss of proliferation, confirming a functional role for the activated EGFR signaling pathway. These results reveal that both the FGFR2 and EGFR family signaling pathways are activated in FGFR2-amplified gastric cancer cell lines to drive cell proliferation and survival. Inhibitors of FGFR2 or Erbb3 signaling may have therapeutic efficacy in the subset of gastric cancers containing FGFR2 amplification. [Cancer Res 2008;68(7):2340–8]

Chronic inflammation has been postulated to play an important role in the pathogenesis of insulin resistance. Inducible nitric oxide synthase (iNOS) has been implicated in many human diseases associated with inflammation. iNOS deficiency was shown to prevent high-fat diet–induced insulin resistance in skeletal muscle but not in the liver. A role for iNOS in fasting hyperglycemia and hepatic insulin resistance, however, remains to be investigated in obesity-related diabetes. To address this issue, we examined the effects of a specific inhibitor for iNOS, l-NIL, in obese diabetic (ob/ob) mice. iNOS expression was increased in the liver of ob/ob mice compared with wild-type mice. Treatment with iNOS inhibitor reversed fasting hyperglycemia with concomitant amelioration of hyperinsulinemia and improved insulin sensitivity in ob/ob mice. iNOS inhibitor also increased the protein expression of insulin receptor substrate (IRS)-1 and -2 1.5- and 2-fold, respectively, and enhanced IRS-1–and IRS-2–mediated insulin signaling in the liver of ob/ob mice. Exposure to NO donor and ectopically expressed iNOS decreased the protein expression of IRS-1 and -2 in cultured hepatocytes. These results suggest that iNOS plays a role in fasting hyperglycemia and contributes to hepatic insulin resistance in ob/ob mice.

Orexin-A and -B are neuropeptides that are implicated in the regulation of vigilance states and energy homeostasis. Orexins are specifically produced by neurons located within the lateral hypothalamic area (LHA), a region implicated in the regulation of feeding behavior. Here, we examined the functional interactions between orexins and anorectic factors [leptin, alpha-melanocyte-stimulating hormone (alpha-MSH) and glucagon-like peptide-1 (GLP-1)] in rats. Intracerebroventricular injection of orexin-A (10 nmol) potently augmented food intake in rats. Neuropeptide Y (NPY) (0.3 nmol) and galanin (3 nmol) also induced a transient increase in food intake. Both NPY- and galanin-induced feeding behaviors were completely inhibited by preadministration of leptin (3 microg), while the same or a higher dose (10 microg) of leptin only partially inhibited orexin-A or -B-induced increase of food intake. Preadministration of anorectic peptides (alpha-MSH and GLP-1), which are shown to be regulated by leptin, abolished NPY-induced feeding; however, orexin-induced feeding was only partially inhibited by these anorectic peptides. These observations suggest that NPY- and galanin-induced increases of feeding involve a leptin-sensitive pathway, while orexin-induced feeding involves both leptin-sensitive and -insensitive pathways.

Phosphoinositides have a pivotal role as precursors to important second messengers and as bona fide signaling and scaffold targeting molecules. Phosphatidylinositol 4-kinases (PtdIns 4-kinases or PI4Ks) are at the apex of the phosphoinsitide cascade. Sequence analysis revealed that mammalian cells contain two type II PtdIns 4-kinase isoforms, now termed PI4KIIalpha and PI4KIIbeta. PI4KIIalpha was cloned first. It is tightly membrane-associated and behaves as an integral membrane protein. In this study, we cloned PI4KIIbeta and compared the two isoforms by monitoring the distribution of endogenous and overexpressed proteins, their modes of association with membranes, their response to growth factor stimulation or Rac-GTP activation, and their kinetic properties. We find that the two kinases have different properties. PI4KIIbeta is primarily cytosolic, and it associates peripherally with plasma membranes, endoplasmic reticulum, and the Golgi. In contrast, PI4KIIalpha is primarily Golgi-associated. Platelet-derived growth factor promotes PI4KIIbeta recruitment to membrane ruffles. This effect is potentially mediated through Rac; overexpression of the constitutively active RacV12 induces membrane ruffling, increases PI4KIIbeta translocation to the plasma membrane, and stimulates its activity. The dominant-negative RacN17 blocks plasma membrane association and inhibits activity. RacV12 does not boost the catalytic activity of PI4KIIalpha further, probably because it is constitutively membrane-bound and already activated. Membrane recruitment is an important mechanism for PI4KIIbeta activation, because microsome-bound PI4KIIbeta is 16 times more active than cytosolic PI4KIIbeta. Membrane-associated PI4KIIbeta is as active as membrane-associated PI4KIIalpha and has essentially identical kinetic properties. We conclude that PI4KIIalpha and PI4KIIbeta may have partially overlapping, but not identical, functions. PI4KIIbeta is activated strongly by membrane association to stimulate phosphatidylinositol 4,5-bisphosphate synthesis at the plasma membrane. These findings provide new insight into how phosphoinositide cascades are propagated in cells.

We have demonstrated involvement of the serotonergic system in orexin-induced behavioral responses in rats. Orexin-A and -B (hypocretin-1 and -2) significantly increased total locomotor activity when administered centrally. They also induced behavioral alterations; increasing grooming, face washing and wet dog shaking in rats. Haloperidol inhibited orexin-induced hyperlocomotion and these behavioral alterations. Serotonin antagonists, ritanserin and metergoline, did not attenuate orexin-induced hyperlocomotion but partly inhibited orexin-induced behavioral alterations. These results suggest that the dopaminergic system might be involved in orexin-induced hyperlocomotion, while both the serotonergic system as well as the dopaminergic system might be involved in orexin-induced behavioral responses.

Genomic characterization of hematologic and solid cancers has revealed recurrent somatic mutations affecting genes encoding the RNA splicing factors SF3B1, U2AF1, SRSF2 and ZRSR2. Recent data reveal that these mutations confer an alteration of function inducing aberrant splicing and rendering spliceosome mutant cells preferentially sensitive to splicing modulation compared with wildtype (WT) cells. Here we describe a novel orally bioavailable small molecule SF3B1 modulator identified through a medicinal chemistry effort aimed at optimizing compounds for preferential lethality in spliceosome mutant cells. H3B-8800 potently binds to WT or mutant SF3b complexes and modulates splicing in in vitro biochemical splicing assays and cellular pharmacodynamic assays. The selectivity of H3B-8800 was confirmed by observing lack of activity in cells expressing SF3B1R1074H, the SF3B1 mutation previously shown to confer resistance to other splicing modulators. Although H3B-8800 binds both WT and mutant SF3B1, it results in preferential lethality of cancer cells expressing SF3B1K700E, SRSF2P95H, or U2AF1S34F mutations compared to WT cells. In animals xenografted with SF3B1K700E knock-in leukemia K562 cells or mice transplanted with Srsf2P95H/MLL-AF9 mouse AML cells, oral H3B-8800 treatment demonstrated splicing modulation and inhibited tumor growth, while no therapeutic impact was seen in WT controls. These data were also evident in patient-derived xenografts (PDX) from patients with CMML where H3B-8800 resulted in a substantial reduction of leukemic burden only in SRSF2-mutant but not in WT CMML PDX models. Additionally, due to the high frequency of U2AF1 mutations in non-small cell lung cancer, H3B-8800 was tested in U2AF1S34F-mutant H441 lung cancer cells. Similar to the results from leukemia models, H3B-8800 demonstrated preferential lethality of U2AF1-mutant cells in vitro and in in vivo orthotopic xenografts at well tolerated doses. RNA-seq of isogenic K562 cells treated with H3B-8800 revealed dose-dependent inhibition of splicing. Although global inhibition of RNA splicing was not observed; H3B-8800 treatment led to preferential intron retention of transcripts with shorter and more GC-rich regions compared to those unaffected by drug. Interestingly, H3B-8800-retained introns commonly disrupted the expression of spliceosomal genes, suggesting that the preferential effect of H3B-8800 on spliceosome mutant cells is due to the dependency of these cells on expression of WT spliceosomal genes. These data identify a novel therapeutic approach with selective lethality in leukemias and lung cancers bearing a spliceosome mutation. Despite the essential nature of splicing, cancer cells without a spliceosome mutation were less sensitive to H3B-8800 compared with potent eradication of mutant counterparts. H3B-8800 is currently undergoing clinical evaluation in patients with MDS, AML, and CMML. Citation Format: Silvia Buonamici, Akihide Yoshimi, Michael Thomas, Michael Seiler, Betty Chan, Benjamin Caleb, Fred Csibi, Rachel Darman, Peter Fekkes, Craig Karr, Gregg Keaney, Amy Kim, Virginia Klimek, Pavan Kumar, Kaiko Kunii, Stanley Chun-Wei Lee, Xiang Liu, Crystal MacKenzie, Carol Meeske, Yoshiharu Mizui, Eric Padron, Eunice Park, Ermira Pazolli, Sudeep Prajapati, Nathalie Rioux, Justin Taylor, John Wang, Markus Warmuth, Huilan Yao, Lihua Yu, Ping Zhu, Omar Abdel-Wahab, Peter Smith. H3B-8800, a novel orally available SF3b modulator, shows preclinical efficacy across spliceosome mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1185. doi:10.1158/1538-7445.AM2017-1185

Higher-density inoculation of fibroblasts into a three-dimensional scaffold should accelerate wound healing after skin implantation. This study attempted to develop tissue-engineered skin with a higher density of fibroblasts. We first attempted to fabricate three-dimensional high-cell-density aggregates (spheroids) of normal human fibroblasts for application to tissue-engineered skin. Our method consisted of rotational shaking with nontreated dishes, decreasing fibroblast-meterial interactions, and augmenting cell-cell interaction. To prompt aggregate formation, the medium was supplemented with insulin, dexamethasone, ascorbic acid, and basic fibroblast growth factors that potentiate secretion of extracellular matrices. Under such improved conditions, fibroblasts were able to form spheroidal aggregates within 24 to 36h of rotational culture. Although the formed aggregates were irregular in shape and were composed of only several cells after 12h, they became almost spheroidal after 24h. The aggregates grew even more round after 36h, and their surfaces became smooth. After 36h of rotational culture, the fibroblast aggregates were collected and reinoculated onto a biodegradable mesh composed of polyglycolic acid coated with collagen. The aggregates were trapped in the material and became attached after 24h. Finally, because transforming growth factor-β3 (TGF-β3) is known to accelerate wound healing, we conducted a semiquantitative analysis of TGF-β3 mRNA in both the fibroblast monolayers (two-dimensional culture) and the aggregates (three-dimensional culture). Analysis of TGF-β3 mRNA expression showed that mRNA expression was greater in the fibroblasts of aggregates than in a monolayer. Therefore, our newly developed dermal graft is expected to accelerate wound healing faster than conventional grafts.

To accelerate wound healing after implantation of tissue-engineered skin products, higher density inoculation of fibroblasts would be important. Therefore, we attempted to develop a tissue-engineered skin with a higher density of fibroblasts by formation of cell aggregates combined with a biodegradable polymer. Our method consisted of rotationally shaking with nontreated dishes, declining fibroblast-material interactions, and augmenting cell–cell interaction. Under the culture condition, normal neonatal human dermal fibroblasts formed spheroidal aggregates. The formation of the fibroblast-aggregates was accelerated by the addition of insulin, dexamethasone, basic fibroblast growth factor, and ascorbic acid to basal medium, and by the control of shaking time. In fibroblast-aggregates formed under a basal medium, the level of gene expression related to wound healing was higher than that of a monolayer when one added the hormones and growth factors. Therefore, tissue-engineered skin products with aggregates and scaffolds would become an artificial skin with potentiality of higher wound healing.

Orexins (orexin-A and -B) are recently identified neuropeptides, which are thought to be implicated in the regulation of feeding behavior. We used a NPY-Y1 receptor specific antagonist, BIBO3304, to examine whether NPY is involved in orexin-induced feeding behavior. Intracerebroventricular administration of orexin-A (10 nmol) induced food intake in rats (food intake for 3 h; vehicle 0.3+/-0.2 g vs. orexin-A 10 nmol, 4.0+/-0.5 g, n=4). Orexin-induced feeding behavior was partially inhibited by prior administration of BIBO3304 (3 h food intake: orexin-A 10 nmol, 4.0+/-0.5 g vs. BIBO3304 (60 microgram) + orexin-A 10 nmol, 2.2+/-0.2 g, n=4). A low dose of BIBO3304 (30 microgram) did not show a significant inhibitory effect. BIBO3457, an inactive enantiomer, used as a negative control, did not show any inhibitory effect on orexin-A-induced feeding behavior. Fos expression was observed in NPY-containing neurons in the arcuate nucleus 1 h after orexin-A (10 nmol) was administered intracerebroventricularly (control 0.3+/-0.08%, orexin-A 10.2+/-0.8%, n=5 rats/group). These observations suggest that NPY is involved in orexin-induced feeding behavior. However, BIBO3304 did not completely abolish the effect of orexin-A. These results suggest that orexin-A elicits feeding behavior partially via the NPY pathway. The NPY system could be the one of downstream pathways by which orexin-A induces feeding behavior. Another pathway may also be involved in orexin-A-induced feeding behavior, because BIBO3304 did not completely abolish orexin-A-induced feeding behavior.

Orexin/hypocretins are recently identified neuropeptides which regulate feeding behaviour. We found orexins increased water intake when administrated intracerebroventricularly to rats. The effect of orexin-A was more potent as compared with orexin-B, suggesting the possible involvement of OX1 receptor. The efficacy of orexin-A was almost comparable with that of angiotensin II, and the effect lasted more than 3 h. Prepro-orexin mRNA level was up-regulated when rats were deprived of water. Orexin-immunoreactive varicose axons were observed in the subfornical organ and area postrema, regions implicated in drinking behaviour. These observations suggest a physiological role for orexin as mediators that regulate drinking behaviour.

Mutations in RNA splicing factors confer an alteration of function and are common in patients with myelodysplastic syndrome (MDS, ~45%), chronic myelomonocytic leukemia (CMML, ~60%), and acute myeloid leukemia (AML) derived from these conditions. Recent data suggest that spliceosome-mutant cells are preferentially sensitive to genetic or pharmacologic splicing modulation compared with wildtype (WT) counterparts. Here, we describe the discovery of H3B-8800, a potent and orally bioavailable modulator of the SF3b complex, and demonstrate efficacy in models of spliceosome mutant myeloid malignancies including a novel xenograft system for CMML. H3B-8800 was identified through a medicinal chemistry approach aimed at identifying compounds with preferential lethality in spliceosome mutant cells. Using a scintillation proximity assay, we demonstrated that H3B-8800 potently binds to SF3b complexes containing either WT or mutant SF3B1 protein. Consistent with this, H3B-8800 showed dose-dependent modulation of splicing in in vitro biochemical splicing assays and cellular pharmacodynamic assays. Selectivity of H3B-8800 for the SF3b complex was confirmed through observing resistance in cells expressing SF3B1R1074H, an SF3B1 mutation previously shown to confer resistance to natural product splicing modulators. In the above biochemical and cellular assays, H3B-8800 affected splicing similarly regardless of spliceosome genotype. However, preferential inhibition of in vitro cell growth was observed in isogenic AML cells with endogenous knock-in of SF3B1K700E or SRSF2P95H mutations compared to WT counterparts. In animals xenografted with SF3B1K700E knock-in K562 cells, oral H3B-8800 treatment demonstrated dose-dependent splicing modulation and inhibited tumor growth, while no therapeutic impact was seen in WT controls. Similarly, anti-leukemic efficacy and improved survival were observed with H3B-8800 treatment in mice transplanted with Srsf2P95H/MLL-AF9 mouse AML cells, a result not seen in Srsf2 WT/MLL-AF9 counterpart leukemias. To understand the preferential effects on spliceosome mutant cells, RNA-seq analysis of isogenic K562 cells treated with H3B-8800 was performed. H3B-8800 induced intron retention and exon skipping, however these effects were not global and introns preferentially retained by H3B-8800 were shorter and more GC-rich compared to those unaffected by drug (Figure A). Interestingly, a substantial number of genes experiencing intron retention with H3B-8800 themselves encoded spliceosome components (Figure B). This suggests that the preferential effect of H3B-8800 on spliceosome mutant cells is due to the exquisite dependency of these cells on normal expression of spliceosome proteins. Next we aimed to understand the therapeutic potential of H3B-8800 in the context of CMML due to the high frequency of SRSF2 mutations and the need for improved outcome in this disorder. To this end, we developed a xenotransplantation model through direct intrafemoral injection of CD34+ cells from CMML patients into "NSGS" mice: a variant of NSG mice that express human IL3, SCF and GM-CSF. We specifically focused on CMML with 200,000 CD34+ cells achieved robust engraftment for all patients (n=7) with rapid lethality (median of 39 days). In vivo H3B-8800 administration substantially reduced leukemic burden in spliceosome-mutant but not spliceosome-WT CMML PDX (Figure C). Furthermore, 2.2-fold reductions in immunophenotypically-defined leukemia initiating cells were seen with H3B-8800 versus vehicle treatment in spliceosome-mutant CMML compared with no change in those mice engrafted with spliceosome-WT CMML. These data identify a novel therapeutic approach with selective lethality in myeloid cells bearing a spliceosome mutation. Despite the essential nature of splicing, CMML/AML cells without a spliceosome mutation were less sensitive to H3B-8800 compared with potent eradication of mutant counterparts. These data demonstrate the therapeutic potential of splicing modulation in spliceosome mutant cancers and H3B-8800 is currently undergoing clinical evaluation in patients with MDS, AML and CMML. Figure. Figure. Disclosures Buonamici: H3 Biomedicine: Employment. Thomas:H3 Biomedicine: Employment. Seiler:H3 Biomedicine: Employment. Chan:H3 Biomedicine: Employment. Caleb:H3 Biomedicine: Employment. Darman:H3 Biomedicine: Employment. Fekkes:H3 Biomedicine: Employment. Karr:H3 Biomedicine: Employment. Liu:H3 Biomedicine: Employment. Meeske:H3 Biomedicine: Employment. Mizui:Eisai: Employment. Pazolli:H3 Biomedicine: Employment. Prajapati:H3 Biomedicine: Employment. Wang:Eisai: Employment. Warmuth:H3 Biomedicine: Employment. Yu:H3 Biomedicine: Employment. Zhu:H3 Biomedicine: Employment. Smith:H3 Biomedicine: Employment.

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.

Optimization of a series of R132H IDH1 inhibitors from a high throughput screen led to the first potent molecules that show robust tumor 2-HG inhibition in a xenograft model. Compound 35 shows good potency in the U87 R132H cell based assay and ∼90% tumor 2-HG inhibition in the corresponding mouse xenograft model following BID dosing. The magnitude and duration of tumor 2-HG inhibition correlates with free plasma concentration.

PDK1 activates AKT suggesting that PDK1 inhibition might suppress tumor development. However, while PDK1 has been investigated intensively as an oncology target, selective inhibitors suitable for in vivo studies have remained elusive. In this study we present the results of in vivo PDK1 inhibition through a universally applicable RNAi approach for functional drug target validation in oncogenic pathway contexts. This approach, which relies on doxycycline-inducible shRNA expression from the Rosa26 locus, is ideal for functional studies of genes like PDK1 where constitutive mouse models lead to strong developmental phenotypes or embryonic lethality. We achieved more than 90% PDK1 knockdown in vivo, a level sufficient to impact physiological functions resulting in hyperinsulinemia and hyperglycemia. This phenotype was reversible on PDK1 reexpression. Unexpectedly, long-term PDK1 knockdown revealed a lack of potent antitumor efficacy in 3 different mouse models of PTEN-deficient cancer. Thus, despite efficient PDK1 knockdown, inhibition of the PI3K pathway was marginal suggesting that PDK1 was not a rate limiting factor. Ex vivo analysis of pharmacological inhibitors revealed that AKT and mTOR inhibitors undergoing clinical development are more effective than PDK1 inhibitors at blocking activated PI3K pathway signaling. Taken together our findings weaken the widely held expectation that PDK1 represents an appealing oncology target. Cancer Res; 71(8); 3052–65. ©2011 AACR.

Heterozygous mutations in several core members of the spliceosome complex have been reported in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). In particular high frequency SF3B1 hotspot mutations, a component of the U2 complex involved in the interaction with the branch point (BP) and recognition of the 3' splice sites (ss) during splicing, have been identified in Refractory Anemia with Ringed Sideroblasts (RARS) a subtype of MDS. Using computational analyses of RNAseq from several cancer types including RARS, we identified that SF3B1 hotspot mutations induce aberrant 3'ss selection by recognizing a cryptic AG located between 15 to 24 nucleotides upstream of the canonical AG. Experimental confirmation of these motif features was performed using minigenes in SF3B1 mutant cells. Furthermore, we discovered that SF3B1 mutant utilized a different BP from that used by SF3B1 wild-type providing novel mechanistic insights into changes in function induced by the hotspot mutations. The induction of aberrant splicing can introduce premature termination codons thus targeting mRNA for degradation by Nonsense Mediated Decay (NMD). We predicted that close to 50% of the aberrantly spliced genes would be subject to NMD and showed (using isogenic Nalm-6 cells engineered by AAV homology to express SF3B1K700E or SF3B1K700K) that several of these genes were downregulated at the transcript and protein levels. These downregulated genes/proteins might be involved in the pathogenesis of SF3B1 mutant cancers. Interestingly, pathway analysis of genes differentially expressed or aberrantly spliced in SF3B1 mutant compared to wild-type in RARS samples identified cell differentiation and epigenetics as the primary misregulated pathways. To study the impact of SF3B1 mutations on differentiation, we used the TF-1 differentiation cell model where erythroid differentiation is induced by treatment with erythropoietin (EPO). EPO treatment, as expected, induced erythroid differentiation in TF-1 cells transduced with SF3B1WT, but a block in erythroid differentiation was observed in TF-1 cells transduced with SF3B1K700E (the most common mutation in MDS and chronic lymphocytic leukemia (CLL)). Intriguingly, SF3B1G742D, which is found mutated in CLL but not MDS, did not block differentiation in this myeloid differentiation model, implying that specific SF3B1 mutations and splicing aberrations have important context dependent effects. Pathway analysis comparing SF3B1K700E vs. SF3B1WT or SF3B1G742D identified several genes involved in heme biosynthesis or downstream of GATA1 to be downregulated (such as, AHSP, ALAS2, CCL5, CD36, EPOR, GP1BB, HBB, HBE1, HBG1, PRG2) in SF3B1K700E cells only. This is consistent with the role of SF3B1K700E in RARS. In our analyses, we also identified that ABCB7 is aberrantly spliced and that the aberrant transcript is subject to NMD, causing downregulation of the canonical transcript and protein. ABCB7 is a mitochondrial transporter important in cellular iron metabolism and in heme production; moreover, partial loss of function mutation in ABCB7 has been identified in X-linked sideroblastic anemia and ataxia, demonstrating an iron overload phenotype in cells with defective ABCB7. Interestingly, when ABCB7 was knocked down in TF-1 cells we observed block in differentiation similar to that observed in SF3B1K700E cells suggesting a link between SF3B1 mutation and ABCB7 levels and impaired differentiation. Taken together, these data suggest that SF3B1 mutations induce aberrant splicing and as a consequence downregulation of several genes that contribute to the block in erythroid differentiation, one of the key biological defects observed in MDS. Disclosures Buonamici: H3 Biomedicine: Employment. Darman:H3 Biomedicine: Employment. Perino:H3 Biomedicine: Employment. Agrawal:H3 Biomedicine: Employment. Peng:H3 Biomedicine: Employment. Seiler:H3 Biomedicine: Employment. Feala:H3 Biomedicine: Employment. Bailey:H3 Biomedicine: Employment. Chan:H3 Biomedicine: Employment. Fekkes:H3 Biomedicine: Employment. Keaney:H3 Biomedicine: Employment. Kumar:H3 Biomedicine: Employment. Kunii:H3 Biomedicine: Employment. Lee:H3 Biomedicine: Employment. Mackenzie:Eisai: Employment. Matijevic:Eisai: Employment. Mizui:H3 Biomedicine: Employment. Myint:Eisai: Employment. Park:H3 Biomedicine: Employment. Pazolli:H3 Biomedicine: Employment. Thomas:H3 Biomedicine: Employment. Wang:H3 Biomedicine: Employment. Warmuth:H3 Biomedicine: Employment. Yu:H3 Biomedicine: Employment. Zhu:H3 Biomedicine: Employment. Furman:Acerta Pharma BV: Research Funding; Gilead: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Speakers Bureau. Ebert:Celgene: Consultancy; H3 Biomedicine: Consultancy; Genoptix: Consultancy, Patents & Royalties. Smith:H3 Biomedicine: Employment.

Myeloid cell leukemia 1 (MCL1) is a member of the BCL2 family of proteins governing the apoptosis pathway and is one of the most frequently amplified genes in cancer. MCL1 overexpression often results in dependence on MCL1 for survival and is linked to resistance to anticancer therapies. However, the development of direct MCL1 inhibitors has proven challenging and new modalities for targeting MCL1 are required. Alternative splicing of MCL1 converts the anti-apoptotic MCL1 long (MCL1L) isoform to the BH3-only MCL1 short (MCL1S) isoform, which has been reported to be pro-apoptotic. Thus, changing MCL1 isoform levels through modulation of RNA splicing may represent an attractive approach to targeting MCL1-amplified cancers. To this end, we tested a collection of small molecule SF3B modulators that impact RNA splicing on MCL1-dependent and MCL1-independent NSCLC cell lines. SF3B modulators induced rapid downregulation of the long form and upregulation of the short- and intron-containing form of MCL1 across models; however, apoptosis was only observed in MCL1-dependent cells. Importantly, SF3B modulators preferentially killed MCL1-dependent cell lines and sensitivity correlated with MCL1 amplification. To dissect the mechanism of SF3B modulator-induced cytotoxicity, we overexpressed either the cDNA for the BH3-only short isoform or the full length isoform of MCL1. Surprisingly, overexpression of MCL1S cDNA had no significant effect on cells by itself and did not sensitize cells to SF3B modulator cytotoxicity. Conversely, MCL1L-specific shRNA knockdown was sufficient to kill MCL1-dependent cells and SF3B modulator cytotoxicity was rescued by expression of MCL1L cDNA. Together, these results argue that MCL1L modulation and not MCL1S upregulation is the effector of SF3B modulator cytotoxicity. In immunocompromised mice bearing MCL1-dependent xenograft models, SF3B1 modulator treatment resulted in significant downregulation of MCL1 levels accompanied by induction of apoptosis and robust efficacy at well-tolerated doses. Moreover, MCL1L cDNA expression in MCL1-dependent models rescued apoptosis induced by SF3B1 modulator treatment. These studies provide proof-of-concept that splicing modulation is an effective strategy for targeting cancers dependent on MCL1. Citation Format: Daniel Aird, Ermira Pazolli, Craig Furman, Linda Lee, Kaiko Kunii, Eun Sun Park, Craig Karr, Betty Chan, Michelle Aicher, Silvia Buonamici, John Yuan Wang, Jacob Feala, Lihua Yu, Markus Warmuth, Peter Smith, Peter Fekkes, Ping Zhu, Baudouin Gerard, Yoshiharu Mizui, Laura Corson. Targeting MCL1-dependent cancers with SF3B splicing modulators. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C8.

Refractory Anemia with Ringed Sideroblasts (RARS), a subtype of Myelodysplatic Syndrome (MDS), occurs with a high frequency of hotspot mutations in HEAT (Huntingtin, Elongation factor 3, protein phosphatase 2A, Targets of rapamycin 1 domains) domains of SF3B1. This protein component of the U2 snRNP complex of the spliceosome is essential in the proper selection and usage of 3' splice sites. RNAseq analysis of MDS and other tumor types in which SF3B1 hotspot mutations have been found show that alternative 3' splice site usage is the predominant cause of RNA transcript aberration. These modifications can result in mRNAs encoding novel peptides, or they can introduce premature termination codons into the pre-mRNA, most likely directing it to the Nonsense Mediated Decay (NMD) pathway for degradation. Using a predictive tool to determine the likelihood of a given aberrant transcript to be targeted for NMD, we determined that nearly 50% of the SF3B1-mutant-associated aberrant transcripts were candidates for degradation. We confirmed this experimentally by treating isogenic Nalm-6 cells (engineered by AAV homology to express SF3B1 K700E or K700K) with or without cycloheximide, an agent known to inhibit translation and RNA degradation by NMD. Investigation of the resulting RNAseq data showed significant rescue of gene expression only for the transcripts predicted to be NMD targets. Ingenuity Pathway Analysis indicated that many of the downregulated genes in SF3B1 mutant samples were involved in differentiation, which has been shown to be dysregulated in MDS. We tested the idea that such modifications in the transcriptome confer selective advantage or impair differentiation in SF3B1 mutant cells. We began by manipulating the expression of ABCB7, one of the genes identified in our RNAseq analysis to be downregulated by aberrant splicing and subsequent NMD. ABCB7 is a mitochondrial transporter important in cellular iron metabolism and, indirectly, in heme production. Additionally, loss of function of ABCB7 is causal in X-linked sideroblastic anemia and has been implicated in RARS MDS. We discovered in our SILAC proteomic analysis that ABCB7 protein was dramatically decreased in K700E SF3B1 Nalm-6 cells relative to K700K Nalm-6, in agreement with our RNAseq analysis. Using doxycycline-inducible shRNA expression, we knocked down ABCB7 mRNA and protein expression in TF-1 erythroblasts. These cells show significant decreases in erythropoeitin (EPO)-induced differentiation when expressing exogenous K700E SF3B1, but not K700R (a very conservative mutation) or WT SF3B1. With direct knock down of ABCB7, we observed a similar phenotype - impairment of EPO-induced differentiation in ABCB7 shRNA-induced cells by Day 7, with no overall decline in cell viability. Interestingly, knock down of SF3B1 expression with shRNA also reduces ABCB7 mRNA. However, it also promotes cell death. This is consistent with the heterozygous nature of SF3B1 hotspot mutations; severe loss of SF3B1 function is deleterious. We propose that hotspot SF3B1 mutants promote aberrant splicing of multiple genes, inducing a general “spliceosomal sickness” in addition to downregulating key genes (e.g. ABCB7) responsible for erythroid differentiation impairment, such as that observed in RARS. Citation Format: Rachel B. Darman, Samantha A. Perino, Michael Seiler, Shouyong Peng, Jacob Feala, Peter Fekkes, Gregg F. Keaney, Kaiko Kunii, Linda Lee, Kian Huat Lim, Yoshiya Oda, Khin Myint, Esther A. Obeng, Ermira Pazolli, Eun Sun Park, John Yuan Wang, Markus Warmuth, Lihua Yu, Ping Zhu, Yoshiharu Mizui, Benjamin L. Ebert, Peter G. Smith, Silvia Buonamici. Mutant SF3B1 downregulates proteins involved in differentiation, including ABCB7. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B125.

Myeloid cell leukemia 1 (MCL1) is a member of the BCL2-family of proteins governing the apoptosis pathway and is one of the most frequently amplified genes in cancer. MCL1 overexpression often results in dependence on MCL1 for survival and is linked to resistance to anticancer therapies. However, the development of direct MCL1 inhibitors has proven challenging and thus far has been unsuccessful. Alternative splicing of MCL1 converts the anti-apoptotic MCL1 long (MCL1-L) isoform to the BH3-only containing MCL1 short (MCL1-S) isoform. As a potential approach for targeting MCL1-dependent cancers, we explored the use of MCL1 splicing modulators. We screened a unique chemical library of compounds that span a range of splicing activities on various substrates in an in vitro assay. Interestingly, we found a subset of general splicing modulators, as well as a subset of SF3B1 inhibitors, that are capable of driving the distinctive alterations in MCL1 splicing that in turn can trigger preferential killing of MCL1-dependent cell lines. The best modulators induce a prominent down-regulation of MCL1-L, up-regulation of MCL1-S, and accumulation of intron-retained MCL1 transcript. Somewhat surprisingly, several additional avenues of investigation pointed to MCL1-L down-regulation rather than MCL1-S up-regulation as the driver of preferential killing of MCL1-dependent cells. This includes the fact that compound-induced cytotoxicity can be rescued by expression of a MCL1-L cDNA and MCL1-L specific shRNA knockdown is sufficient to kill MCL1-dependent cells. On the other hand, overexpression of MCL1-S cDNA had no significant effect on cells and splicing modulators that induced very high levels of MCL1-S mRNA in the absence potent MCL1-L down-regulation exhibit minimal cytotoxicity. Biochemical characterization and understanding of these MCL1 splicing modulators has enabled further optimization of compounds that can induce potent and preferential killing of MCL1-dependent cancer cell lines in vitro. Preliminary studies in mice bearing MCL1-dependent NSCLC xenografts confirmed current lead compounds can indeed induce rapid down-regulation of MCL1-L, induction of apoptosis, and antitumor activity. Collectively these data yield insight into mechanisms of MCL1 splicing modulation that can trigger acute apoptosis in MCL1-dependent cancers and provides support for the idea of using splicing modulators to target difficult-to-drug oncogenic drivers such as MCL1. Citation Format: Eun Sun Park, Michelle Aicher, Daniel Aird, Silvia Buonamici, Betty Chan, Cheryl Eifert, Peter Fekkes, Craig Furman, Baudouin Gerard, Craig Karr, Gregg Keaney, Kaiko Kunii, Linda Lee, Ermira Pazolli, Sudeep Prajapati, Takashi Satoh, Peter Smith, John Yuan Wang, Karen Wang, Markus Warmuth, Lihua Yu, Ping Zhu, Yoshiharu Mizui, Laura B. Corson. Targeting MCL1-dependent cancers through RNA splicing modulation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2941. doi:10.1158/1538-7445.AM2015-2941

Recurrent heterozygous mutations of the spliceosome protein SF3B1 have been identified in myelodysplastic syndromes, chronic lymphocytic leukemia (CLL), breast, pancreatic and skin cancers. SF3B1 is a component of the U2 snRNP complex which binds to the pre-mRNA branch point site and is involved in recognition and stabilization of the spliceosome at the 3′ splice site. To understand the impact of SF3B1 mutations, we compared RNAseq profiles from tumor samples with SF3B1 hotspot mutations (SF3B1-MUT) or wild-type SF3B1 (SF3B1-WT) in breast cancer, melanoma and CLL. This analysis revealed significant increases in the usage of novel alternative splice junctions in SF3B1-MUT samples including selection of alternative 3′ splice sites and less frequently exon skipping. These events induce expression of alternative mRNAs that are translated into novel proteins or aberrant mRNAs that are decayed by cells. A common alternative splicing profile was shared across different hotspot mutations and lineages (e.g. ZDHHC16 and COASY); however, unique alternative splicing profiles were also observed suggesting lineage specific effects. RNAseq analysis of several cell lines with endogenous SF3B1 hotspot mutations confirmed the presence of the same spliced isoforms as observed in tumor samples. To prove that SF3B1-MUT were inducing alternative splicing, transient transfection of several SF3B1 hotspot mutations in 293FT cells induced the expression of the common alternatively spliced genes suggesting functional similarity. Selective shRNA depletion of mutant SF3B1 allele in SF3B1-MUT cells resulted in downregulation of the same splice isoforms. Furthermore, isogenic B-cell lines (NALM-6) expressing the most frequent SF3B1 mutation (K700E) were generated and profiled by RNAseq. As expected, similar alternatively spliced genes were observed in NALM-6 SF3B1-K700E cells exclusively. To investigate the role of nonsense-mediated mRNA decay (NMD) in eliminating aberrant mRNAs induced by SF3B1-MUT, we treated NALM-6 SF3B1-K700E cells with cycloheximide, a translation inhibitor known to inhibit NMD. In the treated samples, expression of several aberrant mRNAs was revealed and some of these transcripts were shown to be downregulated in patient samples. Taken together, these results confirm the association between different SF3B1 hotspot mutations and the presence of novel splice isoforms. We demonstrated that E7107, a potent and selective inhibitor of wild-type SF3B1, also binds and inhibits SF3B1-MUT protein. In addition, E7107 represses the expression of several common aberrant splice mRNA products in SF3B1-MUT cells in vitro and in vivo. When tested in a NALM-6 mouse model, E7107 induced tumor regression and increased the overall survival of animals implanted with NALM-6 SF3B1-K700E cells. These data suggest splicing inhibitors as a promising therapeutic approach for cancer patients carrying SF3B1 mutations. Citation Format: Silvia Buonamici, Kian Huat Lim, Jacob Feala, Eunice Park, Laura Corson, Michelle Aicher, Daniel Aird, Betty Chan, Erik Corcoran, Rachel Darman, Peter Fekkes, Gregg Keaney, Pavan Kumar, Kaiko Kunii, Linda Lee, Xiaoling Puyang, Jose Rodrigues, Anand Selvaraj, Michael Thomas, John Wang, Markus Warmuth, Lihua Yu, Ping Zhu, Peter Smith, Yoshiharu Mizui. SF3B1 mutations induce aberrant mRNA splicing in cancer and confer sensitivity to spliceosome inhibition. [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 2932. doi:10.1158/1538-7445.AM2014-2932