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The PI3K/AKT/mTOR pathway is often activated in lymphoma through alterations in PI3K, PTEN, and B-cell receptor signaling, leading to dysregulation of eIF4A (through its regulators, eIF4B, eIF4G, and PDCD4) and the eIF4F complex. Activation of eIF4F has a direct role in tumorigenesis due to increased synthesis of oncogenes that are dependent on enhanced eIF4A RNA helicase activity for translation. eFT226, which inhibits translation of specific mRNAs by promoting eIF4A1 binding to 5′-untranslated regions (UTR) containing polypurine and/or G-quadruplex recognition motifs, shows potent antiproliferative activity and significant in vivo efficacy against a panel of diffuse large B-cell lymphoma (DLBCL), and Burkitt lymphoma models with ≤1 mg/kg/week intravenous administration. Evaluation of predictive markers of sensitivity or resistance has shown that activation of eIF4A, mediated by mTOR signaling, correlated with eFT226 sensitivity in in vivo xenograft models. Mutation of PTEN is associated with reduced apoptosis in vitro and diminished efficacy in vivo in response to eFT226. In models evaluated with PTEN loss, AKT was stimulated without a corresponding increase in mTOR activation. AKT activation leads to the degradation of PDCD4, which can alter eIF4F complex formation. The association of eFT226 activity with PTEN/PI3K/mTOR pathway regulation of mRNA translation provides a means to identify patient subsets during clinical development.

eFT226: A Selective and Highly Potent Inhibitor of Eukaryotic Initiation Factor 4A (eIF4A), a Novel Approach for the Treatment of Cancer Siegfried H Reich, Peggy A Thompson, Justin T Ernst, Boreth Eam, Nathan P Young, Sarah Fish, Joan Chen, Maria Barrera, Haleigh Howard, Ana Parra, Eric Sung, Jocelyn Staunton, Ivy NJ Hung, Gregory S Parker, Gary G Chiang, Christopher J Wegerski, Andres Nevarez, Jeff Clarine, Samuel Sperry, Alan Xiang, Chinh Tran, Christian Nilewski, Garrick K Packard, Theodore Michels, Paul A Sprengeler, and Kevin R Webster Effector Therapeutics, San Diego, CA Oncoprotein expression is tightly controlled at the level of RNA translation which is largely regulated by the eukaryotic translation initiation factor 4F (eIF4F). eIF4A1, a component of the eIF4F complex, catalyzes the ATP dependent unwinding of RNA duplexes and facilitates 43S ribosome complex scanning within the 5'-untranslated region (UTR). eIF4A1 is required for efficient translation of key oncogenes that contain complex secondary structures within the 5'-UTR. eFT226 is a novel, potent and selective eIF4A small molecule inhibitor with excellent physicochemical and pharmaceutical properties. The design of eFT226 involved ab initio ligand-based methods coupled with small molecule crystal structure analysis. eFT226 inhibits eIF4A1 through a reversible sequence-selective enhancement of eIF4A1 binding to mRNA with specific polypurine motifs within the 5'-UTR. The formation of a stable ternary complex [eIF4A1/eFT226/mRNA] with specific sequence recognition motifs leads to a block in ribosome scanning of select mRNAs. Treatment of lymphoma, AML, breast, colorectal, lung and hepatocellular tumor cell lines with eFT226 led to a dose dependent translational down regulation (IC50 of ~5-20 nM) of key oncogenes that drive tumor cell survival and proliferation (i.e., c-MYC, CCND1, BCL2 and MCL-1). Oncogene down regulation results in potent inhibition of cellular proliferation (GI50 of ~2-30 nM) across a panel of tumor cell lines. Enhanced anti-tumor sensitivity and a rapid induction of apoptosis was observed in hematological cell lines including lymphoma and AML. Inhibition of tumor cell proliferation and survival with eFT226 treatment results in significant antitumor activity in vivo in multiple human tumor models of DLBCL, Burkitt's lymphoma, acute myeloid leukemia (AML), and solid tumors following ≤ 1 mg/kg/week IV administration. eFT226 regulates the protein expression of multiple metabolic markers (i.e. c-MYC, HK2, TXNIP and GLUT1) that result in inhibition of tumor glucose uptake supporting the use of 18F-FDG-PET imaging as a measure of eFT226 target engagement in the clinic. These data demonstrate that eFT226 is a sequence-selective translational repressor of key oncogenic drivers that are essential for tumor cell proliferation and survival and support the clinical development of eFT226 in cancer patients. Citation Format: Siegfried H. Reich, Peggy A. Thompson, Justin T. Ernst, Boreth Eam, Nathan P. Young, Sarah Fish, Joan Chen, Maria Barrera, Haleigh Howard, Ana Parra, Eric Sung, Jocelyn Staunton, Ivy Nj Hung, Gregory S. Parker, Gary G. Chiang, Christopher J. Wegerski, Andres Nevarez, Jeff Clarine, Samuel Sperry, Alan Xiang, Chinh Tran, Christian Nilewski, Garrick K. Packard, Theodore Michels, Paul A. Sprengeler, Kevin R. Webster. eFT226: A selective and highly potent inhibitor of eukaryotic initiation factor 4A (eIF4A), a novel approach for the treatment of cancer [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 DDT02-05.

Mutations in KRAS are among the most common oncogenic lesions across a variety of human cancers. Activation of KRAS directs signaling via the MAPK and PI3K pathways to promote tumor growth. One outcome of enhanced KRAS signaling is the induction of mRNA translation by eIF4A, eIF4E, and eIF4G, which together comprise the eIF4F complex. eIF4A is an RNA helicase that functions to unwind elements in the 5'-untranslated region (UTR) of mRNAs to facilitate scanning of the 40S ribosomal subunit. eFT226 is a highly potent and selective inhibitor of eIF4A that functions by forming a ternary complex between eIF4A, eFT226 and specific polypurine motifs in the 5'-UTR of select mRNAs, thus blocking ribosome scanning and inhibiting mRNA translation. This polypurine sequence motif is highly enriched in the 5'-UTR of eFT226 target genes, many of which are known proto-oncogenes. Translational profiling revealed KRAS to be a target of eFT226 and 5'-UTR sequence analysis of KRAS mRNA identified the polypurine regulatory motif, which imparts sensitivity to eFT226 mediated inhibition of translation. In cell-based reporter assays used to monitor translation, mutation of the KRAS 5'-UTR polypurine motifs resulted in a 10-fold decrease in sensitivity to eFT226 relative to the wild-type sequence. In a cell panel screen for in vitro apoptosis induction by eFT226, the most sensitive models were enriched for cell lines driven by KRAS mutations. Consistent with these results, treatment with eFT226 decreased KRAS protein levels, repressed downstream MAPK signaling, inhibited cell proliferation, and induced apoptosis in a collection of non-small cell lung, colorectal and pancreatic KRAS mutant cancer cell lines. The ability of eFT226 to block tumor cell growth and induce apoptosis is independent of the specific KRAS mutation present (e.g. G12C/V, G13D, Q61H/L) suggesting that eFT226 could be broadly efficacious in treating tumors with activating KRAS mutations. Treatment of KRAS mutant solid tumor xenografts with eFT226 as a monotherapy significantly inhibited tumor growth. Together, these results highlight the ability of eFT226 to inhibit growth and promote apoptosis in KRAS mutant tumors and support the clinical development of eFT226 in KRAS driven tumors. A clinical trial evaluating eFT226 in patients with solid tumor malignancies has initiated. Citation Format: Craig R. Stumpf, Vikas K. Goel, Joan Chen, Jocelyn Staunton, Emily M. Santori, Maria Barrera, Haleigh Howard, Kevin R. Webster, Gary G. Chiang, Peggy A. Thompson. Inhibition of eIF4A by eFT226 blocks KRAS mutant tumor growth [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 1955.

Chimeric antigen receptor (CAR) T cells have shown great promise in treating hematopoietic malignancies, such as leukemia and non-Hodgkin’s lymphoma. The efficacy and durability of CAR T cell therapy have been correlated with higher levels of T stem cell memory (TSCM) and T central memory (TCM) populations. Accordingly, various strategies to enrich these CAR T cell populations are of intense interest. It is well-established that the PI3K/mTOR pathway plays an important role in T cell activation and differentiation, and perturbations in this pathway can enhance T cell memory populations. Work from our group has demonstrated that MNK modifies mTOR signaling and T cell differentiation. Here, we demonstrate that the MNK1/2 inhibitor tomivosertib can substantially increase TCM and TSCM populations in both primary murine and human T cells. Tomivosertib treatment of murine OT-I T cells biases T cell differentiation towards a TCM (CD8+ CD44+ CD62L+) population upon SIINFEKL peptide stimulation in vitro without adverse effects on T cell proliferation, interferon-γ production or cytotoxic function. Similar effects are seen in vivo, where tomivosertib treatment also enriches the TCM cell pool in a SIINFEKL vaccine-induced OT-I adoptive T cell transfer model, which results in increased persistence as demonstrated by a higher memory-recall T cell response upon re-challenge. Furthermore, addition of tomivosertib during production of human CAR (anti-CD19 scFv-4-1BB-CD3ζ) T cells leads to a significantly increased population of TSCM (CD8+ CD45RO CD45RA+ CD27+ CD95+) cells. Based on these data, the combination of tomivosertib with CD19-directed CAR T cells was assessed in the CD19+ Pfeiffer DLBCL model in vivo. Daily oral administration of tomivosertib in combination with CD19-directed CAR T cells results in improved efficacy in comparison to either monotherapy alone, consistent with tomivosertib promoting and maintaining TSCM populations in vivo. Tomivosertib is currently in multiple Phase II clinical trials as a monotherapy or in combination with checkpoint inhibitors. Citation Format: Vikas K. Goel, Rajesh K. Sharma, Jocelyn Staunton, Craig R. Stumpf, Nathan P. Young, Peggy A. Thompson, Gary G. Chiang, Kevin R. Webster. Tomivosertib (eFT508), a potent and highly selective inhibitor of MNK1 and MNK2, enhances CAR T cell activity through modulating T cell differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-068.

An effective and durable T-cell response is a cornerstone of current immunotherapies. We show that eFT508, a potent, selective inhibitor of MNK1 and MNK2, establishes a regulatory program that promotes multiple steps in the cancer immunity cycle including expansion of memory T cells and prevention of T-cell exhaustion. Using OT-I and OT-II transgenic systems, we show that eFT508 shifts the distribution of T cells towards a CD62LhighCD44high central memory (CM) phenotype in both CD4 and CD8 T cells upon activation with SIINFEKL peptide in vitro without adverse effects on T-cell proliferation, interferon-γ production or cytotoxic function. Similar effects are seen in vivo, where eFT508 treatment also enriches the CM T-cell pool in a SIINFEKL vaccine-induced OT-I adoptive T-cell transfer model, which results in increased persistence as demonstrated by a higher memory-recall T-cell response upon rechallenge. In addition, the CM bias elicited by eFT508 remains dominant when combined with agonists of co-stimulatory molecules, such as 4-1BB, OX-40 and GITR, or checkpoint inhibitors, such as PD-1, PD-L1 and CTLA-4, suggesting that eFT508 can affect the rate of T-cell differentiation in these combinations. eFT508 treatment also reduces the expression of exhaustion markers such as PD-1, LAG3 and TIM3, leading to increased cytotoxic T-cell function. eFT508 is currently under evaluation as a single agent in two phase 1/2 clinical trials for patients with advanced solid tumors and patients with advanced lymphoma. In addition, a phase 2 study evaluating eFT508, alone or in combination with avelumab, a PD-L1 immune checkpoint inhibitor, in microsatellite stable relapsed or refractory CRC patients is ongoing. The preclinical studies presented here provide further evidence that eFT508 may combine well with additional immunotherapies beyond checkpoint blockade. Citation Format: Rajesh K. Sharma, Vikas K. Goel, Jocelyn Staunton, Maria Barrera, Ana Parra, Eric Sung, Gary G. Chiang, Kevin R. Webster. eFT508, a potent and highly selective inhibitor of MNK1 and MNK2, regulates T-cell differentiation promoting an antitumor immune response [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 5546.

The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR gamma; NR1C3) plays a central role in adipogenesis and is the molecular target of the thiazolidinedione class of antidiabetic drugs. To overcome the well-known shortcomings of thiazolidinediones, we have identified INT131 (formerly T131 and AMG131) as a potent selective ligand for PPAR gamma that is structurally and pharmacologically distinct from glitazone agonists. In vitro biochemical and cell-based functional assays showed that INT131 mediates a distinct pattern of coregulator recruitment to PPAR gamma. In adipocytes, INT131 showed minimal stimulation of adipocyte differentiation and partially activated PPAR gamma target genes involved in adipogenesis and, at the same time, showed more agonistic activity on another set of target genes that may influence insulin sensitivity directly. These unique properties of INT131 may provide a mechanistic basis for its distinct pharmacological profile. In vivo, increases in glucose tolerance were observed in Zucker (fa/fa) rats following a 14-day oral treatment with INT131. Although the maximal efficacies of INT131 and rosiglitazone were similar with respect to improvements in glucose tolerance, INT131 had less effect on heart and lung weights, weight gain, hemodilution, and plasma volume. Thus, INT131 appears to selectively modulate PPAR gamma responses in an in vivo preclinical model, showing antidiabetic efficacy while exhibiting an improved hemodynamic and cardiovascular adverse effect profile compared to the full agonist rosiglitazone. X-ray crystallography revealed that INT131 interacts with PPAR gamma through a distinct binding mode, forming primarily hydrophobic contacts with the ligand-binding pocket without direct hydrogen-bonding interactions to key residues in helix 12 that are characteristic of full agonists. Mutagenesis studies on Tyr473 in helix 12 demonstrated this residue as essential for rosiglitazone-induced receptor activation, but nonessential for INT131 function in vitro, providing one possible molecular determinant for INT131's distinct pharmacology. INT131 is currently being evaluated in a clinical setting as a therapeutic agent for the treatment of type 2 diabetes.

Dysregulated translation of messenger RNA (mRNA) plays a role in the pathogenesis of multiple solid tumors and hematological malignancies. MNK1 and MNK2 integrate signals from several oncogenic and immune signaling pathways (including RAS, Toll-like receptors and T cell receptor) by phosphorylating eukaryotic initiation factor 4E (eIF4E) and other key effector proteins including hnRNPA1 and PSF. Phosphorylation of these RNA-binding proteins by MNK1 and MNK2 selectively regulates the stability and translation of a subset of cellular mRNA that control tumor/stromal cell signaling, the tumor microenvironment and immune cell function. eFT508 is a potent and highly selective inhibitor of both MNK1 and MNK2. Ribosome profiling has demonstrated that inhibition of MNK1 and MNK2 by eFT508 selectively regulates the translational efficiency and mRNA stability of a subset of genes that include inflammatory cytokines/chemokines, regulators of stress response, and effectors of anti-tumor immune response. Given the importance of MAPK signaling and translational control to immune cell activation and differentiation, the immunological effect of eFT508 was further evaluated in both normal human immune cells in vitro and immunocompetent syngeneic cancer models in vivo. eFT508 treatment of normal donor T cells has no deleterious effect on αCD3/αCD28 stimulated IL-2 production, T cell proliferation or T cell viability. However, eFT508 selectively down regulates the induction of IL-10 and specific immune checkpoint receptors, including PD-1 and LAG3. Further evaluation of the mechanism of translational regulation has shown LAG3 mRNA contains specific sequence elements in the 5’-untranslated region (UTR) that confer sensitivity to eFT508. In addition, IL-10 mRNA is destabilized upon treatment with eFT508 leading to significant inhibition of IL-10 production in activated T cells. Furthermore, eFT508 treatment results in upregulation of MHC class II molecules on tumor cells, macrophage and dendritic cells through an IL-10/MARCH1 dependent mechanism. The in vivo antitumor effect of eFT508 was assessed in the CT26 BALB/C syngeneic tumor model. CT26 mouse tumor cell proliferation and survival are insensitive to eFT508 in vitro. In vivo, daily oral treatment with 1 mg/kg eFT508 results in significant anti-tumor activity, modulation of tumor infiltrating lymphocytes and establishment of immune memory. In addition, combination of eFT508 with either anti-PD-1 or anti-PD-L1 monoclonal antibodies results in marked efficacy, significantly increasing the percentage of responder animals. eFT508 is currently under evaluation in two phase I/II clinical trials for patients with advanced solid tumors and patients with advanced lymphoma respectively. These findings support further clinical evaluation of eFT508 in combination with checkpoint blockade. Citation Format: Kevin R. Webster, Vikas K. Goel, Jocelyn Staunton, Craig R. Stumpf, Rajesh Sharma, Ivy N. Hung, Gregory S. Parker, Jolene Molter, Gary G. Chiang, Christopher J. Wegerski, Samuel Sperry, Vera Huang, Joan Chen, Peggy A. Thompson, Chinh Tran, Justin T. Ernst, Paul A. Sprengeler, Siegfried H. Reich. eFT508, a potent and highly selective inhibitor of MNK1/2 regulates immune checkpoint and cytokine expression promoting anti-tumor immunity [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 596. doi:10.1158/1538-7445.AM2017-596

Purpose: This study was designed to evaluate the potential of eFT508 to selectively regulate key immune signaling pathways and enhance anti-tumor immunity as a monotherapy or in combination with checkpoint blockade in immunocompetent syngeneic cancer models. Methods: eFT508 and its effect on mRNA translation, effector protein production, immune cell signaling and tumor infiltrating lymphocytes was evaluated in vitro using normal human T cells and in vivo utilizing immunocompetent syngeneic models. The mechanism of translational regulation of specific target genes was further evaluated in these model systems. Results: Dysregulated translation of messenger RNA (mRNA) plays a role in the pathogenesis of multiple solid tumors and hematological malignancies. MNK1 and MNK2 integrate signals from several oncogenic and immune signaling pathways (including RAS, p38 and toll-like receptors) by phosphorylating eukaryotic initiation factor 4E (eIF4E) and other key effector proteins including hnRNPA1 and PSF. Phosphorylation of these RNA-binding proteins by MNK1 and MNK2 selectively regulates the stability and translation of a subset of cellular mRNA that control tumor/stromal cell signaling and the tumor microenvironment. eFT508 inhibits both MNK1 and MNK2 through a reversible, ATP-competitive mechanism of action with an IC50 of 2 and 1 nM against MNK1 and MNK2 respectively. eFT508 is highly selective (≥100-fold) for MNK1 and MNK2 relative to over 400 other protein and lipid kinases. Ribosome profiling has demonstrated that inhibition of MNK1 and MNK2 by eFT508 selectively regulates the translational efficiency and mRNA stability of a subset of genes that include inflammatory cytokines/chemokines, regulators of reactive oxygen species (ROS), and effectors of anti-tumor immune response. Given the importance of both RAS signaling and translational control to immune cell function the immunological effect of eFT508 was evaluated in both normal human T cells in vitro and immunocompetent syngeneic cancer models in vivo. eFT508 treatment of normal donor T cells has no deleterious effect on CD3/CD28 activation of IL-2 production, T cell proliferation or on T cell viability. However, eFT508 selectively down regulates the induction of IL-10 and specific immune checkpoint mechanisms. The effect of eFT508 on IL-10 protein production corresponded with reduced mRNA stability. The in vivo antitumor effect of eFT508 was assessed in the CT26 BALB/C syngeneic tumor model. CT26 mouse tumor cell proliferation and survival are insensitive to eFT508 in vitro. In vivo, daily oral treatment with 1 mg/kg eFT508 results in significant anti-tumor activity and establishment of immune memory. In addition, combination of daily oral treatment of 1 mg/kg eFT508 with either anti-PD-1 or anti-PD-L1 monoclonal antibodies increases the number of responder animals and results in synergistic activity that corresponds to the modulation of tumor infiltrating lymphocyte populations. Conclusions: eFT508 is a selective, orally bioavailable small molecule inhibitor of MNK1 and MNK2 that can decrease the production of key immune checkpoint regulators and immunosuppressive cytokines. This novel mechanism of action triggers anti-tumor immune response in immunocompetent syngeneic animal models as a monotherapy and in combination with established immune checkpoint antibodies. eFT508 is currently under evaluation in two phase I clinical trials for patients with advanced solid tumors and patients with advanced lymphoma respectively. These findings support further clinical evaluation of eFT508 in combination with checkpoint blockade. This abstract is also being presented as Poster B29. Citation Format: Kevin R. Webster, Vikas K. Goel, Jocelyn Staunton, Ivy NJ Hung, Gregory S. Parker, Craig R. Stumpf, Jolene Molter, Gary G. Chiang, Christopher J. Wegerski, Samuel Sperry, Joan Chen, Vera Huang, Peggy A. Thompson, Chinh Tran, Justin T. Ernst, Stephen E. Webber, Paul A. Sprengeler, Siegfried H. Reich. eFT508: An oral, potent and highly selective inhibitor of MNK1 and MNK2, promotes anti-tumor immunity as a monotherapy and in combination with immune checkpoint blockade. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr PR11.

Dysregulated translation of messenger RNA (mRNA) plays a role in the pathogenesis of multiple solid tumors and hematological malignancies. MNK1 and MNK2 integrate signals from several oncogenic and immune signaling pathways, including RAS, p38, and Toll-like receptor (TLR) pathways, by phosphorylating eukaryotic initiation factor 4E (eIF4E) and other key effector proteins including hnRNPA1 and PSF. Through phosphorylation of these regulatory proteins MNK1 and MNK2 selectively regulate the stability and translation of a subset of cellular mRNA. eFT508 is a potent, highly selective, and orally bioavailable MNK1 and MNK2 inhibitor. eFT508 has a half-maximal inhibitory concentration (IC50) of 1-2 nM against both MNK isoforms in enzyme assays and inhibits the kinase through a reversible, ATP-competitive mechanism of action. Treatment of tumor cell lines with eFT508 led to a dose-dependent reduction in eIF4E phosphorylation at serine 209 (IC50 = 2-16 nM), consistent with previous findings that phosphorylation of this site is solely dependent upon MNK1/MNK2. In a panel of ~50 hematological cancers, eFT508 showed anti-proliferative activity against multiple DLBCL cell lines. Sensitivity to eFT508 in TMD8, OCI-Ly3 and HBL1 DLBCL cell lines was associated with dose-dependent decreases in production of pro-inflammatory cytokines including TNFα, IL-6, IL-10 and CXCL10. Further evaluation eFT508 mechanism of action demonstrated that decreased TNFα production correlated with a 2-fold decrease in TNFα mRNA half-life. These findings are consistent with MNK1 phosphorylation of specific RNA-binding proteins, eg, hnRNPA1, that regulate the stability and translation of mRNA containing specific AU-rich elements (ARE) in their 3'-untranslated regions (UTR). Pro-inflammatory cytokines are drivers of key hallmarks of cancer including tumor cell survival, migration and invasion, angiogenesis, and immune evasion, while also driving drug resistance. Therefore, eFT508 was tested in vivo in 7 subcutaneous human lymphoma xenograft models. Significant anti-tumor activity was observed in the TMD8 and HBL-1 ABC-DLBCL models, both of which harbor activating MyD88 mutations. In addition, eFT508 combined effectively with components of R-CHOP and with novel targeted agents, including ibrutinib and venetoclax, in human lymphoma models. These results underscore the potential of eFT508 for the treatment of DLBCL. eFT508 has also been characterized in nonclinical safety pharmacology and toxicology studies. Clinical trials in patients with hematological and other malignancies are planned. Disclosures Webster: Effector Therapeutics: Employment. Goel:Effector Therapeutics: Employment. Hung:Effector Therapeutics: Employment. Parker:Effector Therapeutics: Employment. Staunton:Effector Therapeutics: Employment. Neal:Effector Therapeutics: Employment. Molter:Effector Therapeutics: Employment. Chiang:Effector Therapeutics: Employment. Jessen:Effector Therapeutics: Equity Ownership. Wegerski:Effector Therapeutics: Employment. Sperry:Effector Therapeutics: Employment. Huang:Effector Therapeutics: Employment. Chen:Effector Therapeutics: Employment. Thompson:Effector Therapeutics: Employment. Appleman:Effector Therapeutics: Employment. Webber:Effector Therapeutics: Equity Ownership. Sprengeler:Effector Therapeutics: Employment. Reich:Effector Therapeutics: Employment.

MLN0128 is an investigational, orally bioavailable, potent and highly selective, ATP-competitive inhibitor of TORC1/2. A phase 1 study was initiated to evaluate the safety and maximum tolerated dose (MTD) of single-agent MLN0128 at different dose levels and schedules (QW, QD, 3-on/4-off and 5-on/2-off) in patients with advanced nonhematologic tumors. As multiple MTDs were achieved, we used exposure-efficacy modeling to interpret the achieved MTDs in the context of preclinical antitumor efficacy, thus supporting the selection of dosing schedule in the clinic. To this end, we conducted xenograft efficacy studies using the 786-0 renal cell carcinoma xenograft model (PTENmut,VHLdel) in athymic nude mice. MLN0128 was orally administered at a range of different doses under several different schedules (QD, bid QW, 3-on/4-off and 5-on/2-off). Single and repeat-dose PK data was also obtained for a range of different time-points and a linear two-compartment PK model was built. This PK model was used to simulate pharmacokinetic profiles corresponding to the arms of the efficacy studies, for the study duration (504 hours, or 21 days). The average MLN0128 concentrations (Cavg, 0-504) for each arm of the efficacy study were then compared against the tumor growth rate, and found to be broadly proportional, with the exception of the most infrequent (QW) dose schedule, for which efficacy was sub-exposure proportional. A dynamic tumor growth model was found to provide an accurate description of antitumor effect for all schedules, and to predict the schedule effect from the most infrequent schedule. A mathematical analysis demonstrated that the schedule effect occurs due to the saturability of the underlying PK/kill curve, with high infrequent doses resulting in large portions of the PK profile at concentrations where the marginal benefit of additional drug levels approaches zero. A sensitivity analysis conducted on this fitted model found that it was insensitive to tumor growth rate and tumor sensitivity to drug, but not to half-life. This dynamic model will be extended in a translational context, to account for the differences in half-life between mice and humans, as well as to account for the uncertainty in parameter estimation and the human PK variability. Our work thus provides a fundamental basis for the understanding of the PK/efficacy relationship for mTOR inhibitors, which, contrary to some reports in the literature, appears to be mostly AUC-proportional. Highly infrequent dosing was found to be sub-exposure proportional, and our work provides a rational explanation for this effect. Taken together, the approach of constructing a dynamic PK/efficacy model and eventually extending this model into a human setting by adjusting the PK half-life provides a rational basis for the preclinical-to-clinical translation of schedule effects. Citation Format: Mayankbhai Patel, Jocelyn Staunton, Jerome Mettetal, Santhosh Palani, Jeffrey Ecsedy, Chuang Lu, Elly Barry, Eric Westin, Mark Manfredi, Wej Chyi Shyu, Katayoun Jessen, Esha A. Gangolli, Arijit Chakravarty. Preclinical-to-clinical translation of optimal schedule for MLN0128, an investigational novel small-molecule selective mTOR1/2 inhibitor, based on exposure-efficacy modeling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3532. doi:10.1158/1538-7445.AM2013-3532

Background: The mammalian target of rapamycin (mTOR), which comprises two protein complexes, TORC1 and TORC2, regulates tumor cell growth, metabolism and motility. In endometrial cancer, the mTOR pathway is aberrantly activated by constitutive mitogen stimuli, such as FGFR2 and multiple genetic mutations such as PIK3CA, PIK3R1/2, PTEN and LKB1. The activation of multiple signaling pathways, cross-feedback and redundancy of these pathways led us to explore the combination of mTOR kinase inhibitor with chemotherapeutic agents for achieving maximal efficacy in preclinical mouse models of endometrial cancer. Results: INK128 is a potent and selective TORC1/2 inhibitor with excellent drug-like properties currently advancing in clinical development. INK128 inhibits endometrial tumor cell proliferation in vitro and displays tumor growth inhibition in endometrial tumor models. In most instances, the anti-tumor activity of INK128 is cytostatic compared to the cytotoxic effect of taxol and carboplatin. In endometrial tumor cells of diverse genetic backgrounds, combining INK128 with taxol resulted in a synergistic inhibition of tumor cell proliferation, sustained suppression of PI3K/AKT/mTOR pathway activity, skewed cell cycle and an increase in apoptosis. INK128 decreased the expression of anti-apoptotic protein MCL-1 and therefore mechanistically enhanced taxol-triggered apoptosis. In endometrial mouse tumor models with PTEN and FGFR2 mutations, the combination induced marked tumor regression and significantly delayed tumor re-growth upon discontinuation of the treatment compared to taxol treatment alone. The combination therapy was well-tolerated and the enhanced anti-tumor efficacy correlated with mechanism-based inhibition of several TORC1 and TORC2 pharmacodynamic markers and induction of apoptosis in vivo. Additionally, combining INK128 with taxol or carbotaxol resulted in tumor regression in taxol-resistant endometrial tumor models. The opportunity of optimizing dose and schedule combination regimens as well as biomarkers predicting sensitivity/resistance to INK128/taxol combination will be discussed. Conclusion: The combination of mTOR kinase inhibitors with chemotherapeutic agents is a compelling strategy to improve therapeutic outcome and to overcome chemo-resistance for tumor types with aberrant activation of the PI3K/mTOR pathway such as endometrial cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2745. doi:1538-7445.AM2012-2745

Background: Early events of metastatic dissemination arise from the ability of tumor cells to acquire a number of biological changes which allow for their survival in distal microenvironments. Among the early events that initiate disseminated tumor populations include epithelial-to-mesenchymal transition (EMT) and increased potential for invasion that will ultimately lead to colonization of distal sites. The PI3K/mTOR pathway has been linked to clinic-pathalogical features of invasion and metastasis of multiple tumor types. Results: We sought to investigate using both in vitro and in vivo models of metastasis a panel of PI3K/mTOR selective inhibitors, in which INK128, a selective mTOR kinase inhibitor in clinical development was included. Using live-cell time lapse imaging experiments to monitor cell migration and invasion we found that INK128 significantly inhibited motility and velocity of metastatic breast cancer models in wound assays and decreased the ability of tumor cells to migrate through membranes and matrigel. The ability of INK128 to selectively inhibit tumor cell migration in vitro prompted us to assess whether INK128 can prevent tumor dissemination in vivo. In metastatic breast cancer xenograft models we monitored the growth of tumors implanted in mammary fat pads (MFP) and metastatic dissemination by serial noninvasive whole body bioluminescent imaging. We first assessed and characterized the degree of spontaneous metastasis to the lung, liver, auxillary lymph nodes, chest cavity of mice and subsequently the effect of mTOR kinase inihibition on primary tumor growth on the MFP versus overt metastatic dissemination upon daily, oral treatment with INK128. We show that INK128 blocked drastically metastatic spread in a dose-dependent manner without significantly affecting the growth of primary tumors. Interestingly, we found that INK128 did not affect the ability of breast cancer cells to enter into the lungs following an alternative experimental lung metastasis model by lateral tail vein injection but INK128 greatly diminished the capacity of breast cancer cells to subsequently colonize and expand. Conclusion: Our results position mTOR on a metastasis-sustaining axis that engages downstream pathways to support the biological machinery of migration, invasion, and fitness of initiating breast cancer cells during the establishment of thoracic metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2812. doi:1538-7445.AM2012-2812

Background: Multiple agents that inhibit angiogenesis and tumor cell growth via the VEGF and mTOR (TORC1) pathways have been approved for locally advanced or metastatic renal cell carcinoma (RCC) in recent years. Despite this progress, improving overall survival remains a significant clinical challenge for the treatment of this cancer type. The tumorigenesis of certain types of RCC is dominated by genes that play critical role in hypoxia response, including stimulation of neo-angiogenesis. Anti-angiogenic therapies target the tumor microenvironment but provide little direct impact to the tumor cell, allowing the potential for disease progression despite treatment. Approval of allosteric partial TORC1 inhibitors in RCC stimulated interest in pursuing TORC1/2 inhibitors, that block mTOR signaling more potently and comprehensively, alone and in combination with anti-angiogenic agents in preclinical models of RCC. Results: INK128 is an orally bioavailable, potent and selective ATP site mTOR kinase inhibitor (TORC1/2) that has entered clinical development. INK128 inhibited the phosphorylation of S6 and 4EBP1, downstream substrates of TORC1, and AKT and NRDG1, downstream substrate of TORC2 in RCC cell lines. TORC1/2 pathway inhibition correlated with potent and complete blockade of cell proliferation. In vivo, daily treatment of INK128 as single agent suppressed tumor growth in multiple mouse RCC xenograft models. Pharmacodynamic analysis demonstrated that INK128 inhibits both TORC1 and TORC2 pathways in vivo. INK128 did not alter the tumor-associated microvasculature density despite significant decrease of VEGF supply by lowering the capacity of protein translation of the tumor cells. This prompted us to explore the potential of the combination of INK128 with anti-angiogenic agents to maximize anti-tumor activity by targeting both tumor microenvironment and tumor cells directly. Co-treatment of INK128 with sorafenib resulted in much enhanced activity in RCC tumor models but did not achieve sufficient tolerability. We next employed an alternating dosing strategy of INK128 and sorafenib to improve tolerability. An optimized dose/schedule sequence of both agents caused sustained inhibition of tumor-associated angiogenesis, suppression of mTOR pathway resulting into marked blockade of tumor growth with significant induction of apoptosis and acceptable tolerability. Conclusion: The sequential, yet potent blockade of both mTOR and VEGF pathways obtained by combining INK128 with sorafenib acts synergistically in inducing cell death and tumor regression in preclinical renal cancer models. Adaptive combination of receptor tyrosine kinase-based anti-angiogenic agents with mTOR kinase inhibitors currently in development may offer a safe and effective therapeutic strategy for renal cancer patients who fail to respond to standard of care therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3753. doi:1538-7445.AM2012-3753

Background: The PI3K pathway is one of the most frequently dysregulated pathways in human cancer. Activating mutations in the PIK3CA gene, encoding the p110 catalytic subunit of PI3K, have been identified as a major mechanism of inducing oncogenic PI3K signaling. The high frequency of PIK3CA mutations suggests that PI3K inhibitors may have therapeutic utility in genetically defined tumor populations. The discovery of isoform-selective PI3K inhibitors has proven to be difficult due to the highly homologous nature of all PI3K isoforms and structurally related kinases. PI3Kα-selective inhibitors may permit more potent inhibition of PI3Kα while minimizing side effects and permitting more alternatives for combination therapy relative to non-selective class I PI3K (pan-PI3K) pathway inhibitors, many of which have entered clinical development. Results: Through structure-guided drug design we have discovered INK1117, a novel, potent and selective PI3K inhibitor with good oral bioavailability. INK1117 potently inhibits PI3K and demonstrates a greater than 100-fold selectivity relative to other class I PI3K family members and mTOR as well as a high degree of selectivity against a large panel of protein kinases. INK1117 blocks proliferation of tumor cell lines bearing PIK3CA mutations, and inhibits cellular phosphorylation and activity of AKT. However, INK1117 shows much less activity in PTEN-deficient tumor cells, which typically display constitutive PI3K pathway activation independent of PI3Kα. Daily, oral administration of INK1117 potently inhibits tumor growth in xenograft models bearing PIK3CA oncogenic mutations and, comparable to in vitro studies, INK1117 was not efficacious in tumor models with PTEN and/or KRAS mutations. In contrast to pan-PI3K inhibitors, INK1117 does not induce significant glucose elevation in glucose unchallenged or challenged rodents while levels of circulating Insulin is mildly elevated. The absence of hyperglycemia but presence of moderate hyperinsulinemia in mice treated with INK1117 is in line with results obtained using genetically engineered PI3K deficient mice. Additionally, INK1117 does not significantly impair B and T cell function in vitro and in vivo. INK1117 in combination with targeted agents such as lapatinib or trastuzumab, displayed enhanced activity both in vitro and in vivo. Our preclinical data show that PI3K isoform-selective kinase inhibitors as a single agent can provide comparable or superior efficacy than panPI3K inhibitors in tumors with PIK3CA mutation with better tolerability. The combination of PI3K inhibitors with other oncogene-targeted drugs revealed further improved anti-tumor efficacy. Conclusion: Selective targeting of PI3K combined with companion diagnostic presents a novel therapeutic strategy for patients with PIK3CA mutated cancer types. INK1117 is a potent and orally efficacious PI3Kα-selective kinase inhibitor currently in phase 1 clinical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A171.

Background: The mammalian target of rapamycin (mTOR) comprises two protein complexes, TORC1 and TORC2, which together regulate cell growth, metabolism, angiogenesis, and cell survival. Because TORC1 and TORC2 play a crucial role in several pathways that are frequently dysregulated in human cancer, the TORC1/2 kinase inhibitors provide a promising class of anti-cancer agents. Activation of multiple genomic and signaling pathways, cross-feedback, pathway redundancy and the capacity for compensatory adaptive response in cancer suggest that the optimal therapeutic effect of mTOR inhibitors will be better exploited by the strategy of rational combination therapies. Results: INK128 is a potent and selective TORC1/2 inhibitor with excellent drug-like properties currently in multiple phase 1 studies. Daily, oral administration of INK128 inhibits tumor growth in multiple xenograft models with predicted pharmacokinetic/pharmacodynamic relationship. In most instances the anti-tumor effect of INK128 is cytostatic compared to the cytotoxic effect of chemotherapeutic agents such as Taxol. To enhance the anti-tumor activity of INK128, we investigated the combination of INK128 with cytotoxic agents such as conventional chemo drugs or apoptosis inducing agents. Treatment of tumors with the cytotoxic agents such as Taxol often leads to activation of the PI3K/AKT/mTOR pathway, which limits the anti-tumor activity of Taxol and may eventually lead to drug resistance. Additionally, Taxol resistance mechanisms include the over-expression of members of the Bcl-2 family of proteins such as MCL-1 and BCL-XL, whose expression is regulated by TORC1 and their function possibly by TORC2. Therefore, there is a strong scientific rationale to investigate the combination of a TORC1/2 inhibitor and Taxol. To this end, we have investigated the preclinical efficacy of INK128 in combination with Taxol in vitro and in vivo. Combining INK128 with Taxol resulted in a synergistic inhibition of tumor cell proliferation and the suppression of PI3K/AKT/mTOR signaling pathways in various cancer types. In most instances alterations in cell cycle and an increase in apoptosis was observed. INK128 decreases the expression of anti-apoptotic protein Mcl-1 and therefore resensitizes Taxol-resistant tumor cells to Mcl-1-dependent build up. In various xenograft models, the combination therapies were well-tolerated and displayed enhanced anti-tumor efficacy, inhibited pharmacodynamic markers, and induced apoptosis. Combination of INK128 with a small molecule inhibitor of Bcl-2 (ABT-263) also led to significant increase of apoptosis in tumor cells but not in normal cells in vitro and enhanced anti-tumor activity in mouse tumor models in vivo. Conclusion: In summary, our data show a potential benefit for combining INK128 with cytotoxic agents. INK128 is currently in a Taxol combination phase Ib study in patients with advanced solid malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A172.

Objective: Preclinical characterization and evaluation of INK128 alone and in combination with anti-angiogenic agents in models of renal cell carcinoma. Background: mTOR kinase comprises two distinct multi-protein complexes, TORC1 and TORC2, which together regulate processes critical for cell growth and survival. TORC1 regulates protein translation of cell cycle regulators, angiogenic factors, and factors that control cell motility. TORC2 regulates pathways involved in cell metabolism, survival and motility. Through rational drug design we have identified INK128, a potent and selective small molecule, active-site kinase inhibitor of mTOR with excellent drug-like properties. Activity seen with temsirolimus and everolimus, pharmaceutical derivatives of rapamycin and allosteric partial inhibitors of TORC1, provide clinical proof-of-concept for targeting mTOR for cancer therapy. TORC1 is upstream of HIF1/2 and TORC1 and TORC2 are downstream of the VEGF pathways implicated in the tumorigenesis of metastatic renal cell carcinoma (RCC). We have investigated INK128 in preclinical in vitro and in vivo models of renal cell carcinoma (RCC) and compared its activity to other standard of care agents commonly used to treat this disease. Results: INK128 selectively inhibited phosphorylation of S6 and 4EBP1, downstream substrates of TORC1, as well as phosphorylation of AKT downstream of TORC2 in vitro and in vivo. INK128 demonstrated very potent inhibition of tumor cell proliferation and induction of G1 cell cycle arrest in vitro. In mouse tumor models, INK128, rapamycin, avastin and sorafenib displayed anti-tumor efficacy; however, they differ in the mechanisms underlying the anti-tumor activity. INK128 inhibits phosphorylation of AKT, PRAS40, NDRG1, S6 and 4EBP1; rapamycin only inhibits S6 phosphorylation and induces phosphorylation of AKT; sorafenib activates several pathway components that are downstream of TORC1/2. INK128 and rapamycin suppressed tumor growth by directly inhibiting tumor cell proliferation; however neither had much impact on tumor-associated angiogenesis. In contrast, sorafenib and avastin suppressed tumor growth by potently inhibiting tumor angiogenesis. The activity of the combination of INK128 with sorafenib or avastin yielded sustained tumor regression by targeting tumor cells and the microenvironment. Conclusion: INK128 offers a novel approach for the treatment of renal cell carcinoma by targeting TORC1/2 signaling. Additionally, the mechanism of action of INK128 is different than current therapies and may not display cross resistance with current standard of care agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4486. doi:10.1158/1538-7445.AM2011-4486

Objective: Discovery, characterization and preclinical evaluation of PI3Kalpha (PI3Kα) selective small molecule kinase inhibitors for the treatment of solid tumors. Background: The PI3K pathway is one of the most frequently dysregulated pathways in human cancer. Activating mutations in the PIK3CA gene, encoding the p110 catalytic subunit of PI3Kα, have been identified as a major mechanism of inducing oncogenic PI3K signaling. The high frequency of PIK3CA mutations suggests that PI3Kα inhibitors may have therapeutic utility in genetically defined tumor populations. The discovery of isoform-selective PI3Kα inhibitors has proven to be difficult due to the highly homologous nature of all PI3K isoforms and structurally related kinases. Multiple non-selective class I PI3K (pan-PI3K) inhibitors have entered clinical development. PI3Kα-selective inhibitors may permit more potent inhibition of PI3Kα while minimizing side effects and permitting more alternatives for combination therapy relative to pan-PI3K pathway inhibitors. Results: Through structure-guided drug design we have discovered INK1117, a novel, potent and selective PI3Kα inhibitor with good oral bioavailability. INK1117 potently inhibits PI3Kα and demonstrates a greater than 100-fold selectivity relative to other class I PI3K family members and mTOR as well as a high degree of selectivity against a large panel of protein kinases. INK1117 blocks proliferation of tumor cell lines bearing PIK3CA mutations, and inhibits cellular phosphorylation and activity of AKT. However, INK1117 shows much less activity in PTEN-deficient tumor cells, which typically display constitutive PI3K pathway activation independent of PI3Kα. INK1117 blocks VEGF signaling and angiogenesis in vitro and in vivo. Daily, oral administration of INK1117 potently inhibits tumor growth in xenograft models bearing PIK3CA oncogenic mutations and, comparable to in vitro studies, INK1117 was not efficacious in tumor models with PTEN and/or KRAS mutations. In contrast to pan-PI3K inhibitors, INK1117 does not appear to impair glucose homeostasis or insulin response in glucose/insulin tolerance tests in rodents. Additionally, INK1117 does not significantly impair B and T cell function in vitro and in vivo. These two factors support the hypothesis that INK1117 may be better tolerated than pan-PI3K inhibitors, particularly when used in combination with other agents. Conclusion: INK1117 is a potent and orally efficacious PI3Kα isoform-selective kinase inhibitor with excellent drug-like properties. Selectively targeting PI3Kα offers a new therapeutic approach for the treatment of cancers with PIK3CA activation and preclinical data suggest that isoform-selective inhibitors may provide equivalent efficacy to pan-PI3K inhibitors in selected tumors with a more favorable safety profile. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4501. doi:10.1158/1538-7445.AM2011-4501

mTOR kinase operates via two distinct multi-protein complexes, TORC1 and TORC2, which together regulate growth, metabolism, angiogenesis and survival. Because the PI3K/Akt/mTOR pathway integrates nutrient and hormonal signaling and is frequently dysregulated in human cancer, the mTOR kinase has become an important target for oncology drug development. However, multiple genomic and signaling pathways are often simultaneously activated in cancer, and pathway redundancy and compensatory feedback can blunt the activity of even the most potent anticancer agents. Thus, it is important to study the anti-tumor efficacy of potential anticancer agents, both alone and in combination with cytotoxic and targeted agents, to achieve the optimal therapeutic effect. Through rational drug design we have discovered INK128, a potent and selective TORC1/2 inhibitor with outstanding drug-like properties. INK128 inhibits both the phosphorylation of S6 and 4EBP1, downstream substrates of TORC1, and selectively inhibits AKT phosphorylation at Ser473, the downstream substrate of TORC2, both in vitro and in vivo. Potent inhibition was also observed in cell lines resistant to rapamycin and PanPI3K inhibitors. Daily, oral administration of INK128 inhibited angiogenesis and tumor growth in multiple xenograft models with predicted PK/PD relationship. We have studied INK128 in combination with chemotherapeutic agents as well as with molecular targeted agents both in vitro and in vivo tumor models. In most cases, INK128 demonstrated a synergistically enhanced inhibition of tumor growth and suppression of respective signaling pathways. Additionally, induction of apoptosis was observed only when INK128 was used in combination, suggesting apoptosis as a potential contributor to the observed synergy. A subset of the agents that displayed synergy when combined with INK128 in vitro were evaluated in a number of xenograft models. The combinations were well-tolerated and displayed both enhanced anti-tumor efficacy and enhanced inhibition of key pharmacodynamic markers. In summary, INK128 offers a compelling approach to the treatment of cancer either as a single agent or in combination with other anti-cancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4496.

Pharmacokinetic / pharmacodynamic (PK/PD) modeling is a valuable strategy to achieve target inhibition as well as predictable and meaningful therapeutic efficacy. The mammalian target of rapamycin (mTOR) comprises two protein complexes, TORC1 and TORC2, which together regulate cell growth, metabolism, angiogenesis, and cell survival. Because TORC1 and TORC2 play a crucial role in several pathways that are frequently dysregulated in human cancer, the mTOR kinase is a compelling target for oncology drug development. Through rational drug design we have identified INK128, a potent and selective small molecule, ATP-competitive, active-site TORC1/2 kinase inhibitor with excellent drug-like properties. Inhibition of phosphorylation of S6 and 4EBP1, downstream markers of TORC1 signaling, was selected for PD analysis in peripheral blood cells (PBCs), skin tissue, and tumor tissue biopsy in mice xenograft tumor models. Time- and dose-dependent inhibition of S6 and 4EBP1 was demonstrated in PBCs by phospho-flow (FACS) analysis. Immunohistochemistry and immunoblot analysis demonstrated a correlation between S6 and 4EBP1 inhibition in tumors or skin tissue and antitumor effect. Additionally, site-selective inhibition of AKT phosphorylation at Ser473, the downstream substrate of TORC2, was also demonstrated in tumors and skin biopsies in mouse xenograft models. Our results demonstrate that daily, oral administration of INK128 selectively inhibits PI3K/AKT/mTOR signaling at the level of TORC1/2, and show that INK128 inhibits growth, and in some cases induces regression, of various tumor xenograft models. Results from these studies display a clear pharmacokinetic and pharmacodynamic relationship. Moreover, the activity of several of these downstream markers can be reproducibly measured in human peripheral blood cells and may permit development of a PK/PD model that might assist to predict PBC and skin tissue PD marker inhibition time-profiles in patients. In summary, INK128 presents a compelling, biomarker-guided approach for the treatment of a variety of cancer by targeting TORC1/2 signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1668.