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The application and integration of molecular profiling technologies create novel opportunities for personalized medicine. Here, we introduce the Tumor Profiler Study, an observational trial combining a prospective diagnostic approach to assess the relevance of in-depth tumor profiling to support clinical decision-making with an exploratory approach to improve the biological understanding of the disease.

Purpose: The PI3K/Akt/mTOR pathway is an attractive target in HER2-positive breast cancer that is refractory to anti-HER2 therapy. The hypothesis is that the suppression of this pathway results in sensitization to anti-HER2 agents. However, this combinatorial strategy has not been comprehensively tested in models of trastuzumab and lapatinib resistance. Experimental Design: We analyzed in vitro cell viability and induction of apoptosis in five different cell lines resistant to trastuzumab and lapatinib. Inhibition of HER2/HER3 phosphorylation, PI3K/Akt/mTOR, and extracellular signal-regulated kinase (ERK) signaling pathways was evaluated by Western blotting. Tumor growth inhibition after treatment with lapatinib, INK-128, or the combination of both agents was evaluated in three different animal models: two cell-based xenograft models refractory to both trastuzumab and lapatinib and a xenograft derived from a patient who relapsed on trastuzumab-based therapy. Results: The addition of lapatinib to INK-128 prevented both HER2 and HER3 phosphorylation induced by INK-128, resulting in inhibition of both PI3K/Akt/mTOR and ERK pathways. This dual blockade produced synergistic induction of cell death in five different HER2-positive cell lines resistant to trastuzumab and lapatinib. In vivo, both cell line–based and patient-derived xenografts showed exquisite sensitivity to the antitumor activity of the combination of lapatinib and INK-128, which resulted in durable tumor shrinkage and exhibited no signs of toxicity in these models. Conclusions: The simultaneous blockade of both PI3K/Akt/mTOR and ERK pathways obtained by combining lapatinib with INK-128 acts synergistically in inducing cell death and tumor regression in breast cancer models refractory to anti-HER2 therapy. Clin Cancer Res; 18(9); 2603–12. ©2012 AACR.

The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth and cancer. However, the downstream translationally regulated nodes of gene expression that may direct cancer development are poorly characterized. Using ribosome profiling, we uncover specialized translation of the prostate cancer genome by oncogenic mTOR signalling, revealing a remarkably specific repertoire of genes involved in cell proliferation, metabolism and invasion. We extend these findings by functionally characterizing a class of translationally controlled pro-invasion messenger RNAs that we show direct prostate cancer invasion and metastasis downstream of oncogenic mTOR signalling. Furthermore, we develop a clinically relevant ATP site inhibitor of mTOR, INK128, which reprograms this gene expression signature with therapeutic benefit for prostate cancer metastasis, for which there is presently no cure. Together, these findings extend our understanding of how the ‘cancerous’ translation machinery steers specific cancer cell behaviours, including metastasis, and may be therapeutically targeted.

mTOR activation leads to enhanced survival signaling in acute myeloid leukemia (AML) cells. The active-site mTOR inhibitors (asTORi) represent a promising new approach to targeting mTOR in AKT/mTOR signaling. MLN0128 is an orally-administered, second-generation asTORi, currently in clinical development. We examined the anti-leukemic effects and the mechanisms of action of MLN0128 in AML cell lines and primary samples, with a particular focus on its effect in AML stem/progenitor cells. MLN0128 inhibited cell proliferation and induced apoptosis in AML by attenuating the activity of mTOR complex 1 and 2. Using time-of-flight mass cytometry, we demonstrated that MLN0128 selectively targeted and functionally inhibited AML stem/progenitor cells with high AKT/mTOR signaling activity. Using the reverse-phase protein array technique, we measured expression and phosphorylation changes in response to MLN0128 in 151 proteins from 24 primary AML samples and identified several pro-survival pathways that antagonize MLN0128-induced cellular stress. A combined blockade of AKT/mTOR signaling and these pro-survival pathways facilitated AML cell killing. Our findings provide a rationale for the clinical use of MLN0128 to target AML and AML stem/progenitor cells, and support the use of combinatorial multi-targeted approaches in AML therapy.

Originally discovered as oncogene-associated lipid kinases more than 15 years ago, the family of phosphoinositide 3-kinase (PI 3-K) enzymes has recently emerged as an important therapeutic target in human pathophysiology. After more than a decade with only a few inhibitors with low activity, poor isoform or kinase selectivity or unacceptable pharmacological and toxicological profiles, a variety of PI 3-K inhibitors with pan-isoform activity and isoform-selective inhibition have recently been identified. A growing excitement surrounds the success of signal transduction modifiers in cancer therapy, as well as in the therapy of other diseases. In combination with additional genetic evidence implicating the PI 3-K pathway in human disease, the search for more selective and more drug-like PI 3-K inhibitors has been reinvigorated. As a consequence, many biotech and pharmaceutical companies have established PI 3-K pathway drug development programmes. With the help of important insights provided by genetic knocko...

Activating mutants of ras are among the most frequently found genetic alterations in human cancers. Therefore, Ras appears to be an attractive target for therapeutic intervention using gene transfer. The protein kinase Raf-1 acts as a direct downstream effector of Ras and is involved in Ras-induced cellular transformation. Using the NIH3T3 fibroblast-derived tumor cell line PEJ, which expresses oncogenic Ha-rasG12V, we analyzed whether dominant negative mutants of Raf-1 can inhibit Ras-mediated transformation. Retroviral gene transfer was used to stably transduce PEJ cells with three different dominant negative mutants of Raf-1. This resulted in reversion of the transformed phenotype in vitro as evidenced by an increase in contact inhibition and reduced anchorage-independent growth. However, tumor formation in nude mice was significantly delayed only by one of these mutants. Therefore, dominant negative mutants of the oncoprotein Myc, which is known to synergize with Raf-1 in tumor formation, were transduced into PEJ cells expressing a dominant negative Raf mutant. This leads to killing of the cells. These results indicate that although interference with Ras-induced transformation using dominant negative mutants of Raf is feasible and effective in vitro using retroviral vectors, an additional block (e.g., that of Myc) is necessary to kill PEJ cells. These results also indicate that interference with Ras-dependent signaling is not sufficient for inhibition of tumor formation of PEJ cells in vivo.

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase whose activity contributes to leukemia proliferation and survival. Compounds targeting the mTOR active site inhibit rapamycin-resistant functions and have enhanced anti-cancer activity in mouse models. MLN0128 (formerly known as INK128) is a novel, orally active mTOR kinase inhibitor currently in clinical development. Here we evaluated MLN0128 in preclinical models of B-cell acute lymphoblastic leukemia (B-ALL). MLN0128 suppressed proliferation of B-ALL cell lines in vitro and reduced colony formation by primary human leukemia cells from adult and pediatric B-ALL patients. MLN0128 also boosted the efficacy of dasatinib in Philadelphia Chromosome-positive (Ph+) specimens. In a syngeneic mouse model of lymphoid BCR-ABL+ disease, daily oral dosing of MLN0128 rapidly cleared leukemic outgrowth. In primary xenografts of Ph+ B-ALL specimens, MLN0128 significantly enhanced the efficacy of dasatinib. In non-Ph B-ALL xenografts, single agent MLN0128 had a cytostatic effect that was most pronounced in mice with low disease burden. In all in vivo models, MLN0128 was well tolerated and did not suppress endogenous bone marrow proliferation. These findings support the rationale for clinical testing of MLN0128 in both adult and pediatric B-ALL and provide insight towards optimizing therapeutic efficacy of mTOR kinase inhibitors.

The interactions between the bone marrow (BM) microenvironment and acute myeloid leukemia (AML) is known to promote survival of AML cells. In this study, we used reverse phase-protein array (RPPA) technology to measure changes in multiple proteins induced by stroma in leukemic cells. We then investigated the potential of an mTOR kinase inhibitor, PP242, to disrupt leukemia/stroma interactions, and examined the effects of PP242 in vivo using a mouse model. Using RPPA, we confirmed that multiple survival signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), were up-regulated in primary AML cells cocultured with stroma. PP242 effectively induced apoptosis in primary samples cultured with or without stroma. Mechanistically, PP242 attenuated the activities of mTORC1 and mTORC2, sequentially inhibited phosphorylated AKT, S6K, and 4EBP1, and concurrently suppressed chemokine receptor CXCR4 expression in primary leukemic cells and in stromal cells cultured alone or cocultured with leukemic cells. In the in vivo leukemia mouse model, PP242 inhibited mTOR signaling in leukemic cells and demonstrated a greater antileukemia effect than rapamycin. Our findings indicate that disrupting mTOR/AKT signaling with a selective mTOR kinase inhibitor can effectively target leukemic cells within the BM microenvironment.

Aberrant activation of the mammalian target of rapamycin (mTOR) signaling plays an important role in breast cancer progression and represents a potential therapeutic target for breast cancer. In this study, we report the impact of the investigational drug MLN0128, a potent and selective small molecule active-site TORC1/2 kinase inhibitor, on tumor growth and metastasis using human breast cancer xenograft models. We assessed in vitro antiproliferative activity of MLN0128 in a panel of breast cancer cell lines. We next evaluated the impact of MLN0128 on tumor growth, angiogenesis and metastasis using mammary fat pad xenograft models of a non-VEGF (ML20) and a VEGF-driven (MV165) MCF-7 sublines harboring PIK3CA mutations. MLN0128 potently inhibited cell proliferation in various breast cancer cell lines harboring PIK3CA (IC(50): 1.5-53 nM), PTEN (IC(50): 1-149 nM), KRAS, and/or BRAF mutations (IC(50): 13-162 nM), and in human endothelial cells (IC(50): 33-40 nM) in vitro. In vivo, MLN0128 decreased primary tumor growth significantly in both non-VEGF (ML20; p = 0.05) and VEGF-driven MCF-7 (MV165; p = 0.014) xenograft models. MLN0128 decreased the phosphorylation of Akt, S6, 4E-BP1, and NDRG1 in both models. In contrast, rapamycin increased Akt activity and failed to reduce the phosphorylation of 4E-BP1, PRAS40, and NDRG1. VEGF-induced lung metastasis in MV165 is inhibited by MLN0128 and rapamycin. In conclusion, MLN0128 inhibits TORC1/2-dependent signaling in preclinical models of breast cancer. MLN0128 appears to be superior in blocking mTORC1/2 signaling in contrast to rapamycin. Our findings support the clinical research of MLN0128 in patients with breast cancer and metastasis.

Several phosphoinositide 3-kinase (PI3K) inhibitors are in the clinic and many more are in preclinical development. CAL-101, a selective inhibitor of the PI3Kδ isoform, has shown remarkable success in certain hematologic malignancies. Although PI3Kδ signaling plays a central role in lymphocyte biology, the degree of single-agent therapeutic activity of CAL-101 during early-phase development has been somewhat unexpected. CAL-101 works in part by blocking signals from the microenvironment that normally sustain leukemia and lymphoma cells in a protective niche. As PI3Ks enter the arena of molecular-targeted therapies, CAL-101 provides proof of principle that isoform-selective compounds can be effective in selected cancer types and patient populations. Significance: A key question is whether compounds targeting a single PI3K catalytic isoform can provide meaningful single agent efficacy in cancer cells that express multiple isoforms. Clinical studies of the drug CAL-101 have provided a significant advance by showing that selective targeting of PI3Kδ achieves efficacy in chronic lymphocytic leukemia, in part through targeting the tumor microenvironment. Cancer Discovery; 1(7); 562–72. ©2011 AACR.

Class I phosphoinositide 3 kinase (PI3K) δ is a promising therapeutic target for rheumatoid arthritis (RA) because of its contribution to leukocyte biology. However, its contribution in fibroblasts has not been studied as a mechanism that contributes to efficacy. We investigated the expression and function of PI3Kδ in synovium and cultured fibroblast-like synoviocytes (FLS). Immunohistochemistry demonstrated that PI3Kδ is highly expressed in RA synovium, especially in the synovial lining. Using quantitative PCR and Western blot analysis, we found that PI3Kδ mRNA and protein expression is higher in RA than in osteoarthritis (OA) synovium. PI3Kδ was also expressed in cultured FLS, along with PI3Kα and PI3Kβ, whereas PI3Kγ was not detectable. PI3Kδ mRNA expression was selectively induced by inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) but not by growth factors platelet-derived growth factor (PDGF) and transforming growth factor β (TGFβ). The use of inhibitors that block individual PI3K isoforms, including the novel selective PI3Kδ inhibitor INK007, showed that PI3Kδ is required for PDGF- and TNF-induced Akt activation. PI3Kδ inhibition also diminished PDGF-mediated synoviocyte growth and sensitized cells to H 2 O 2 -induced apoptosis. These data are the first documentation of increased PI3Kδ expression in both RA synovium and cultured synoviocytes. Furthermore, these are the first data demonstrating that PI3Kδ is a major regulator of PDGF-mediated fibroblast growth and survival via Akt. Thus, targeting PI3Kδ in RA could modulate synoviocyte function via anti-inflammatory and disease-altering mechanisms.

The multiple roles of PI3Kδ (p110δ) and PI3Kγ (p110γ) in various immune cells have encouraged the development of small-molecule inhibitors of both PI3K isoforms, alone or in combination, for the treatment of immune-mediated inflamatory diseases. Recent findings suggest a previously unrecognized interdependent cooperativity between p110δ and p110γ, if not all class I PI3K isoforms, expressed and activated in leukocytes and endothelium. For example, the activity of p110δ and p110γ in combination appears to be necessary for mediating efficient (velocity and direction) trafficking of immune competent cells to sites of inflammation. This chapter will focus on the emerging evidence of the dynamic interplay of p110δ and p110γ supporting the hypothesis that dual-blockade of both, p110δ and p110γ, presents a unique therapeutic opportunity in that pharmacological inhibition of the two PI3K isoforms simultaneously may yield superior clinical results in the treatment of a variety of complex immune-mediated inflammatory diseases (Fig. 1).

Targeting the mammalian target of rapamycin (mTOR) protein is a promising strategy for cancer therapy. The mTOR kinase functions in two complexes, TORC1 (target of rapamycin complex-1) and TORC2 (target of rapamycin complex-2); however, neither of these complexes is fully inhibited by the allosteric inhibitor rapamycin or its analogs. We compared rapamycin with PP242, an inhibitor of the active site of mTOR in both TORC1 and TORC2 (hereafter referred to as TORC1/2), in models of acute leukemia harboring the Philadelphia chromosome (Ph) translocation. We demonstrate that PP242, but not rapamycin, causes death of mouse and human leukemia cells. In vivo, PP242 delays leukemia onset and augments the effects of the current front-line tyrosine kinase inhibitors more effectively than does rapamycin. Unexpectedly, PP242 has much weaker effects than rapamycin on the proliferation and function of normal lymphocytes. PI-103, a less selective TORC1/2 inhibitor that also targets phosphoinositide 3-kinase (PI3K), is more immunosuppressive than PP242. These findings establish that Ph(+) transformed cells are more sensitive than normal lymphocytes to selective TORC1/2 inhibitors and support the development of such inhibitors for leukemia therapy.

We describe novel, cell-permeable, and bioavailable salicylic acid derivatives that are potent and selective inhibitors of GLEPP1/protein-tyrosine phosphatase φ. Two previously described GLEPP1 substrates, paxillin and Syk, are both required for cytoskeletal rearrangement and cellular motility of leukocytes in chemotaxis. We show here that GLEPP1 inhibitors prevent dephosphorylation of Syk1 and paxillin in resting cells and block primary human monocyte and mouse bone marrow-derived macrophage chemotaxis in a gradient of monocyte chemotactic protein-1. In mice, the GLEPP1 inhibitors also reduce thioglycolate-induced peritoneal chemotaxis of neutrophils, lymphocytes, and macrophages. In murine disease models, the GLEPP1 inhibitors significantly reduce severity of contact hypersensitivity, a model for allergic dermatitis, and dextran sulfate sodium-induced ulcerative colitis, a model for inflammatory bowel disease. Taken together, our data provide confirmation that GLEPP1 plays an important role in controlling chemotaxis of multiple types of leukocytes and that pharmacological inhibition of this phosphatase may have therapeutic use.

Background— The role of inflammation at all stages of the atherosclerotic process has become an active area of investigation, and there is a notable quest for novel and innovative drugs for the treatment of atherosclerosis. The lipid kinase phosphoinositide 3-kinase-γ (PI3Kγ) is thought to be a key player in various inflammatory, autoimmune, and allergic processes. These properties and the expression of PI3Kγ in the cardiovascular system suggest that PI3Kγ plays a role in atherosclerosis. Methods and Results— Here, we demonstrate that a specific PI3Kγ inhibitor (AS605240) is effective in murine models of established atherosclerosis. Intraperitoneal administration of AS605240 (10 mg/kg daily) significantly decreased early atherosclerotic lesions in apolipoprotein E–deficient mice and attenuated advanced atherosclerosis in low-density lipoprotein receptor–deficient mice. Furthermore, PI3Kγ levels were elevated in both human and murine atherosclerotic lesions. Comparison of low-density lipoprotein receptor–deficient mice transplanted with wild-type or PI3Kγ-deficient bone marrow demonstrated that functional PI3Kγ in the hematopoietic lineage is required for atherosclerotic progression. Alleviation of atherosclerosis by targeting of PI3Kγ activity was accompanied by decreased macrophage and T-cell infiltration, as well as increased plaque stabilization. Conclusions— These data identify PI3Kγ as a new target in atherosclerosis with the potential to modulate multiple stages of atherosclerotic lesion formation, such as fatty streak constitution, cellular composition, and final fibrous cap establishment.

Abnormal mTOR activation leads to enhanced survival signaling in acute myeloid leukemia (AML) cells. The active site mTOR kinase inhibitors (TOR-KI) represent a promising new approach to target the PI3K/AKT/mTOR pathway. In a previous study, we used a synthetic TOR-KI PP242 to demonstrate that disrupting AKT/mTOR signaling with this class of compounds can effectively target leukemic cells under conditions mimicking bone marrow microenvironment[1]. In the current study, we focused on the inhibitory mechanisms in rare and heterogeneous AML stem cells and on the cellular antagonistic responses after mTORC1/2 blockage using MLN0128, a synthetic TOR-KI currently in clinical development. We found that MLN0128 inhibited cell proliferation and induced apoptosis in AML cells by attenuating mTOR complex 1 and 2 (mTORC1/2) activities, and showed higher anti-leukemia potency than PP242. Using high-throughput time-of-flight mass cytometry (CyTOF)[2], combined with advanced computational analytic software Spanning-Tree Progression of Density-Normalized Events (SPADE)[3] and visualization t-Distribution Stochastic Neighbor Embedding (viSNE)[4], we identified AML subsets co-expressing multiple stem cell markers with high AKT/mTOR activity. MLN0128 selectively targets mTORC1/2 downstream signaling in these cells with minimal effects on other pathways, and lacks the effect on non- leukemic stem cells (LSC). The inhibitory effect of MLN0128 on LSC is positively correlated to that of a number of co-expressing stem cell molecules, and to the baseline expression of and the degree of SCF activation on AKT/mTOR signaling. Using the reverse phase protein array (RPPA) technique, we studied expression and phosphorylation changes in 151 proteins from 24 primary AML samples in response to MLN0128, and revealed several pro-survival pathways may protect from MLN0128-induced cellular stress. We identified four druggable targets that were significantly triggered by MLN0128 inhibition: PARP (that can be targeted by PARP inhibitor olaparib), HDAC3 (vorinostat), p-SRC (dasatinib), and STAT5 (ruxolitinib). Blocking AKT/mTOR signaling and these pro-survival cascades triggered complex responses in AML cell lines and primary AML samples, resulting in an overall increase in apoptosis of AML cells compared to single target inhibition. Among the four combinations, MLN0128 and vorinostat elicited the best responses. In summary, our findings indicate that blocking AKT/mTOR signaling with the mTORC1/2 inhibitor MLN0128 causes growth inhibition and apoptosis in AML cells. High-throughput CyTOF technology revealed that MLN0128 selectively targets primary AML stem cells with high AKT/mTOR activity. RPPA mapping multiple intracellular signaling pathways in primary AML cells in response to mTORC1/2 inhibition allowed us to identify compensatory activation of actionable pro-survival pathways. Combinatorial multi-targeted approaches demonstrated various responses to MLN0128 in AML samples, suggesting that personalized treatment selections will be needed in individual AML cases. Finally, utilization of techniques such as CyTOF or RPPA may assist in profiling intra-cellular and inter-patient variability of responses to targeted agents which likely stem from multiple genetic and epigenetic events within individual tumor, and serve as tools for selection of individualized targeted therapies in AML and other malignancies. Reference 1. Zeng, Z.H., et al., Targeting of mTORC1/2 by the mTOR kinase inhibitor PP242 induces apoptosis in AML cells under conditions mimicking the bone marrow microenvironment. Blood, 2012. 120(13): p. 2679-2689. 2. Bendall, S.C., et al., Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum. Science, 2011. 332(6030): p. 687-696. 3. Qiu, P., et al., Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE. Nature Biotechnology, 2011. 29(10): p. 886-U181. 4. Amir el, A.D., et al., viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat Biotechnol, 2013. 31(6): p. 545-52. Disclosures No relevant conflicts of interest to declare.

Growth factor stimulated receptor tyrosine kinases activate a protein kinase cascade via the serine/threonine protein kinase Raf-1. Direct upstream activators of Raf-1 are Ras and Src. This study shows that MEK1, the direct downstream effector of Raf-1, can also stimulate Raf-1 kinase activity by a positive feedback loop. Activated MEK1 mediates hyperphosphorylation of the amino terminal regulatory as well as of the carboxy terminal catalytic domain of Raf-1. The hyperphosphorylation of Raf-1 correlates with a change in the tryptic phosphopeptide pattern only at the carboxy terminus of Raf-1 and an increase in Raf-1 kinase activity. MEK1-mediated Raf-1 activation is inhibited by co-expression of the MAPK specific phosphatase MKP-1 indicating that the MEK1 effect is exerted through a MAPK dependent pathway. Stimulation of Raf-1 activity by MEK1 is independent of Ras, Src and tyrosine phosphorylation of Raf-1. MEK1 can however synergize with Ras and leads to further increase of the Raf-1 kinase activity. Thus, MEK1 can mediate activation of Raf-1 by a novel positive feedback mechanism which allows fast signal amplification and could prolong activation of Raf-1.

Purpose: The PI3K/Akt/mTOR pathway is an attractive target in HER2 positive breast cancer refractory to anti-HER2 therapy. The hypothesis is that suppression of this pathway results in sensitization to anti-HER2 agents. However, this combinatorial strategy has not been comprehensively tested in models of trastuzumab and lapatinib resistance. Experimental Design: We analyzed in vitro cell viability and induction of apoptosis in five different cell lines resistant to trastuzumab and lapatinib. Inhibition of HER2/HER3 phosphorylation, PI3K/Akt/mTOR and ERK signaling pathways was evaluated by western blot. Tumor growth inhibition following treatment with lapatinib, INK-128 or the combination of both agents was evaluated in three different animal models: two cell-based xenograft models refractory to both trastuzumab and lapatinib and a xenograft derived from a patient who relapsed to trastuzumab-based therapy. Results: The addition of lapatinib to INK-128 prevented both HER2 and HER3 phosphorylation induced by INK-128, resulting in inhibition of both PI3K/Akt/mTOR and ERK pathways. This dual blockade synergistically enhanced cell death in five different HER2 positive cell lines resistant to trastuzumab and lapatinib. In vivo, both cell line-based and patient-derived xenografts showed exquisite sensitivity to the antitumor activity of the combination of lapatinib and INK-128, causing durable tumor shrinkage in the absence of any sign of toxicity. Conclusions: The simultaneous blockade of PI3K/Akt/mTOR and ERK pathways achieved by combining lapatinib with INK-128 acts synergistically in inducing cell death and tumor regression in breast cancer refractory to anti-HER2 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 1811. doi:1538-7445.AM2012-1811

Background: INK128 is an orally bioavailable potent and selective ATP site kinase inhibitor of mammalian target of rapamycin (mTOR) complexes TORC1 and TORC2 that play a crucial role in regulating tumor cell growth, metabolism and motility. INK128 is structurally and mechanistically distinct from rapamycin and rapalogs. mTOR activity is frequently dysregulated in cancer by constitutive mitogen stimuli or oncogenic mutations upstream of TORC1/2. TORC1/2 kinase inhibitors are a promising class of novel anti-cancer agents. Methods: Adult patients (pts) with histologically confirmed advanced solid tumors were enrolled in a typical 3+3 dose escalation Phase I study evaluating 3 intermittent schedules: QW (once weekly), QDx3d QW (3 days on 4 days off), and QDx5d QW (5 days on 2 days off). Safety, maximum tolerated dose (MTD), pharmacokinetics (PK) and preliminary antitumor activity were evaluated. Pharmacodynamic (PD) endpoints were evaluated in surrogate (peripheral blood cells [PBCs], skin) and tumor tissues for the phosphorylation of TORC1-dependent markers (4EBP1/S6), and TORC2-dependent markers (AKT/PRAS40). Results: To date, 52 pts have been treated in 3 intermittent dosing regimens; 21 in 6 cohorts ranging 7-40 mg QW, 21 in 5 cohorts ranging 6-20 mg QDx3d QW, and 10 in 3 cohorts ranging 7-13 mg QDx5d QW. Dose limiting toxicities of Grade (G) 3 asthenia and G3 mucositis were reported in the 40 mg QW, 20 mg QDx3d QW, and 13 mg QDx5d QW cohorts. The MTD for intermittent dosing has not been reached. All adverse events (AEs) reported have been reversible. The most common (≥20%, n=35) AEs considered possibly related to INK128 reported in all 3 dosing regimens included nausea (51%), hyperglycemia (37%), mucosal inflammation (29%), rash (23%), asthenia (23%), vomiting (26%), and diarrhea (20%). The majority of AEs considered possibly related to INK128 in any regimen were G1 or 2. The only reported G ≥3 AE (≥5%) possibly related to INK128 in 3 regimens was lymphopenia (6%). Comparison across the 3 dosing regimens showed consistent and dose-dependent PK. INK128 was rapidly absorbed with Tmax ranging from 0.5 to 4 h and a mean elimination plasma t1/2 of 8 h. The plasma exposures (Cmax and AUC0-24) following oral doses suggest dose-linear plasma PK. Decreases in p4EBP1 levels were seen in PBCs in all dosing regimens. Skin biopsies showed 60∼100% inhibition of TORC1 (p4EBP1 and pS6) and TORC2 (pPRAS40). Preliminary anti-tumor activity was seen in pts with lung and renal cancer. Conclusions: INK128 is well-tolerated at the doses and schedules tested and shows high oral bioavailability and dose-linear PK across all dosing regimens evaluated with exposures in the range of predicted biological activity. PD biomarker changes are consistent with inhibition of TORC1 and TORC2. Preliminary anti-tumor activity is encouraging in this ongoing study. 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 CT-02. doi:1538-7445.AM2012-CT-02

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: The PI3K-AKT-mTOR pathway is commonly activated in breast cancers due to the amplification of receptor tyrosine kinase (HER2), the mutation of PIK3CA or the loss of expression/function of PTEN. About 20% of HER2+ breast cancer patients have PIK3CA mutation, which has resulted in a trend toward shorter progression-free survival with trastuzumab-based therapy. Resistance (intrinsic or acquired) to trastuzumab is a significant clinical challenge, partly due to the overriding activation of PI3K-AKT-mTOR pathway. The PI3K pathway activation leads to stimulation of the key growth and proliferation regulatory kinase, mTOR which can be blocked by allosteric inhibitors (rapalogous) or by ATP-comptetive mTOR kinase inhibitor (TORC1/2). Purpose: We investigated the effects of INK128; a potent and selective TORC1/2 kinase inhibitor alone or in conjunction with trastuzumab in HER2 amplified breast tumor cell lines. We also sought to identify the molecular response determinants of mTOR kinase inhibitor predicting potent activity in HER2+/trastuzumab-resistant cell lines. Experimental Design: Here, we used the HER2 amplified trastuzumab-sensitive breast tumor cell line (BT474), the trastuzumab-resistant breast tumor cell line (BT474HR) and HER2 amplified/PIK3CA mutated breast tumor cell lines (HCC1954, UACC893). Results: We demonstrate that 1) HER2+ cells are sensitive to INK128 (IC50 ∼40nM), 2) INK128 is also effective in trastuzumab-resistant and PIK3CA mutaed/HER2+ breast tumor cells (IC50 ∼20 and 20-30nM respectively), 3) INK128 is mechanistically distinct from rapalogous by a more potent and comprehensive inhibition of PI3K-AKT-mTOR signaling network: a) avoids S6K inhibition-mediated feedback activation of PI3K-AKT and b) abrogates 4EBP1 phosphorylation; 4) ligand-induced HIF1α accumulation is inhibited by prior treatment of INK128, and 5) the combination of trastuzumab plus INK128 more effectively blocked PI3K-AKT-mTOR signaling and HIF1α accumulation both in parental and trastuzumab-resistant cells. Conclusion: Our results suggest that i) therapeutic targeting of PI3K-AKT-mTOR signaling should be effective in abrogating resistance to trastuzumab therapy in HER2+ breast cancer, ii) combination of trastuzumab and INK128 is a promising treatment approach for HER2 overexpressed as well as trastuzumab-resistant breast cancer patients and iii) dual TORC1/2 inhibitor also mitigates the feedback activation of AKT caused by selective inhibition of mTORC1, and because of this, INK128 may yield greater therapeutic benefit in breast cancer patients. 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 2240. doi:1538-7445.AM2012-2240

Background: INK128 is an orally bioavailable potent and selective ATP site kinase inhibitor of mammalian target of rapamycin (mTOR) complexes TORC1 and TORC2 that play a crucial role in regulating tumor cell growth, metabolism, and motility. INK128 is structurally and mechanistically distinct from rapamycin and rapalogs. mTOR activity is frequently dysregulated in human cancer by constitutive mitogen stimuli or oncogenic mutations upstream of TORC1 and TORC2. TORC1/2 kinase inhibitors are a promising class of novel anti-cancer agents. Methods: Adult patients (pts) with histologically confirmed advanced solid tumors were enrolled in a typical 3+3 dose escalation Phase I study to determine the safety, maximum tolerated dose (MTD), pharmacokinetics (PK), pharmacodynamics (PD) and preliminary antitumor activity. PD endpoints were evaluated in surrogate (peripheral blood cells [PBCs] and skin) and tumor tissues for the phosphorylation of TORC1-dependent markers (S6/4EBP1), and TORC2-dependent markers (AKT/PRAS40). Results: A total of 25 pts received daily (QD) doses of INK128 in 4 cohorts at 2 mg (n=3), 4 mg (n=7), 6 mg (n=7), and 7 mg (n=8). Dose-limiting toxicities (DLTs) were Grade (G) 3 hyperglycemia, G3 rash, and G4 anemia. The MTD was 6 mg QD with a DLT of G3 rash in 1 out of 6 pts. All AEs reported to date have been reversible. The most common (>20%, n=25) AEs considered possibly related to INK128 included hyperglycemia (88%), rash (52%), nausea (36%), pruritus (36%), diarrhea (32%), dysgeusia (32%), mucosal inflammation (32%), asthenia (24%), blood creatinine increased (24%), decreased appetite (24%), fatigue (24%), and vomiting (24%). The most common G ≥3 AEs (≥10%; n=25) considered possibly related to INK128 were rash (28%) and hyperglycemia (16%). Hyperglycemia was generally well controlled with metformin. INK128 was rapidly absorbed with Tmax of 1 to 4 h with high oral bioavailability. The mean elimination t1/2 ranged from 7 to 11 h and the mean steady-state plasma concentrations (Cmax, ss) ranged from 52 nM to 210 nM across 4 dose levels. Comparisons of plasma exposures (Cmax and AUC0-24 following oral doses suggest dose-linear plasma PK with limited inter-pt variability. PD biomarker measurements in PBCs, skin, and tumor showed dose-dependent TORC1/2 inhibition as evidenced by decreases in p4EBP1, pS6, and PRAS40. Preliminary anti-tumor activity was seen in 24% and 12% of pts experiencing stable disease for ≥4 and ≥6 cycles, respectively. Conclusions: INK128 dosed QD at its MTD of 6 mg has an acceptable tolerability and safety profile in pts with advanced malignancies. INK128 shows high oral bioavailability and dose-linear PK with exposures in the range of predicted biological activity. PD biomarker changes are consistent with inhibition of TORC1 and TORC2. Preliminary antitumor activity is encouraging in this ongoing study. 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 5588. doi:1538-7445.AM2012-5588

Introduction: Recent understanding of the biology of Triple Negative (TN) tumor cells has resulted in the recognition of following molecular targets for the development of novel therapeutics, (1) cell surface receptor tyrosine kinases (RTKs, e.g. EGFR, IGFR, c-MET, FGFR), (2) intracellular signaling pathway(s) (PI3K-AKT-mTOR, RAS-MAPK-ERK), and (3) DNA-damage (chemo- or radiotherapy) repair pathway. INK128 is an orally bioavailable, potent and selective ATP site kinase inhibitor of mTOR complexes TORC1 and TORC2 that together play a crucial role in regulating tumor cell growth, metabolism and motility. INK128 is structurally and mechanistically distinct from allosteric rapamycin-based mTOR inhibitors such as RAD001. Methods: We tested effects of INK128 (alone or in combination with carboplatin and ABT888) on, (a) cell survival/proliferation, (b) cell signaling marker(s) of proliferation, (c) fibronectin-mediated migration, (d) fibronectin-directed matrigel-invasion, and (e) clonogenic survival (3D-ON-TOP assay) in different TNBT cell lines with high expression of EGFR (MDA-MB468, SUM149), BRCA mutations (HCC1937, SUM149) no expression of PTEN protein (HCC70, HCC1937, MDA-MB468, SUM149), PIK3CA mutation (BT20) and RAS/RAF mutation (MDA-MB231). Results: Data showed that, (1) although the range of EC50s for INK128 varied from 50 nM-500 nM in different TNBT cells, the PTEN-null cells exhibited favorable EC50s compared to the rest of the cell lines (with exception to HCC1937), (2) inhibition of phosphorylation of AKT (S473), S6RP, and 4EBP1 was observed following INK128 treatment (100 nM and 200 nM) as early as 6 hours, (3) treatment with 200 nM INK128 for 24 hours differentially blocked pERK in PTEN-null MDA-MB468 as compared to MDA-MB231 cells, (4) INK128 treatment (100 nM) inhibited migration of different TNBT cells (as compared to 2 uM RAD001, allosteric, partial TORC1 inhibitor)) and the inhibition was differentially pronounced in PTEN-null TNBT cells lines, (5) INK128 treatment (100 nM) also blocked invasion of MDA-MB468 cells, and (6) INK128 dose-dependently blocked clonogenic growth of TNBT cells. This effect was potentiated in the presence of ABT888 (10uM; small molecule PARP inhibitor) plus carboplatin (10uM; chemotherapeutic agent). Conclusion: INK128 (alone or in combination with carboplatin and ABT888) has anti-proliferative effect on TNBT cells. INK128 has anti-migratory and anti-invasive effects on TNBT cells. Interestingly, the anti-migratory effect of INK128 was most prominent in PTEN-null TNBT cells. We are currently pursuing studies to test the effect of INK128 on the endothelial cells, the results of which will be presented in the meeting. 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 2230. doi:1538-7445.AM2012-2230

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

Abstract 1815 Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 (Raptor) and TORC2 (Rictor). TORC1 leads to the phosphorylation of p70S6 kinase and 4E- BP1, while TORC2 regulates phosphorylation of Akt and other kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin and its analogues have not shown significant activity in MM, likely due to the lack of inhibition of TORC2. In this study, we dissected the baseline activity of the PI3K/Akt/mTOR pathway TORC1/2 in MM cell lines with different genetic abnormalities. Methods: Eight different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, immunochemistry, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. In vivo homing was checked by in vivo flow. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: Raptor (TORC1) and Rictor (TORC2) knockdowns led to significant inhibition of proliferation of MM cells even in the presence of bone marrow stromal cells, this effect was also accompanied by inactivation of p-Akt, p-rS6 and p-4EBP1. We used INK128, a dual and selective TORC1/2 kinase inhibitor with similar effects to Raptor plus Rictor knockdown. We examined the protein expression levels of both mTOR complex and their downstream effectors in MM plasma cells from patients and cell lines. mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all samples. We showed that dual TORC1/2 inhibition is much more active than TORC1 inhibition alone (rapamycin) even in the presence of cytokines or stromal cells. INK128 induced cell cycle arrest, autophagy and apoptosis in cell lines and primary plasma cells even in the presence of bone marrow stromal cells (BMSCs). INK128 also showed a significant effect inhibiting cell adhesion in our in vivo homing model. Oral daily treatment with INK128 highly decreased the percentage of CD138+ tumor plasma cells in mice implanted with MM cells and reduced the levels of p-Akt and p-4EBP. These results suggest that potent and complete blockade of mTOR as part of TORC1 and TORC2 is potential therapeutic strategy to induce cell cycle arrest, apoptosis and disruption of MM cells interaction with the BM microenvironment. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Liu: Intellikine: Employment. Roccaro:Roche: Research Funding. Rommel:Intellikine: Employment. Ghobrial:Celgene: Consultancy; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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

MAPK pathway mutations are frequent in colorectal cancer (CRC) and associated with resistance to standard therapies. This provides a rationale for the use of targeted therapies against the MAPK pathway. However, the response of CRC cells to MEK inhibitors varies depending on coexisting mutations in both MAPK and PI3K pathways. As a matter of fact, dual inhibition of MEK and PI3K or AKT in mouse models of CRC has proven effective due to aberrations in both pathways. Inhibition of TORC1, a shared downstream effector of the MAPK and PI3K pathways, was described to be partly responsible for the efficacy of the dual blockade. In order to explore additional mechanisms involved in the superior antitumor activity of combined MAPK and PI3K pathway suppression we directly targeted TORC1/2 by active-site kinase inhibitor of mTOR, in addition to MEK, in our cell models. We tested the antiproliferative activity of INK128, a selective and potent TORC1/2 inhibitor, in combination with a MEK inhibitor, PD-0325901, in a panel of colon cancer cell lines with diverse alterations of the MAPK and PI3K pathways. PD-0325901 treatment effectively inhibited ERK phosphorylation, which resulted in decreased phosphorylation of Rb and reduced expression of E2F in all the cell lines tested. In CRC cell lines harboring sole mutations in the MAPK pathway, we observed sensitivity to PD-0325901 that was not further enhanced when combining with INK-128. In contrast, CRC cell lines harboring concurrent mutations of both pathways exhibited a striking sensitivity to dual ERK and TORC1/2 blockade associated with an enhanced arrest in cell cycle and, above all, a concomitant increase in apoptosis. Inhibition of TORC1 and TORC2 by INK128 could not be further enhanced by MEK inhibition in any of the cell lines tested. These data suggest that, besides complete suppression of both pathways, additional mechanisms must be at work to explain the observed enhanced cell death following dual suppression of PI3K and MEK. Interestingly, we observed differential regulation of key components of the cell cycle in response to dual blockade. We are currently investigating whether this specific biochemical behaviour plays a causative role in the observed phenotype. In summary, dual inhibition of TORC1/2 and MEK has potential for effective antitumor activity in CRCs with dysregulation of both signaling cascades. A better understanding of the molecular mechanisms responsible for the enhanced efficacy of this therapeutic combination will help defining the best biomarkers that need to be assessed in phase I combination trials. 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 653. doi:10.1158/1538-7445.AM2011-653

Abstract 133 Introduction: Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment such as cytokines and growth factors, nutrients and stresses to regulate multiple cellular processes, including translation, autophagy, metabolism, growth, motility and survival. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 and TORC2. Activation of TORC1 leads to the phosphorylation of p70S6 kinase and 4E-BP1, while activation of TORC2 regulates phosphorylation of Akt and other AGC kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin analogues such as RAD001 and CCI-779 have been tested in clinical trials in MM. Their efficacy as single agents is modest, but when used in combination, they show higher responses. However, total inhibition of Akt and 4E-BP1 signaling requires inactivation of both complexes TORC1 and TORC2. Consequently, there is a need for novel inhibitors that can target mTOR in both signaling complexes. In this study we have evaluated the role of TORC1 and TORC2 in MM and the activity and mechanism of action of INK128, a novel, potent, selective and orally active small molecule TORC1/2 kinase inhibitor. Methods: Nine different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: To examine activation of the mTOR pathway in MM, we performed kinase activity assays and protein analyses of mTOR complexes and its downstream targets in nine MM cell lines. We found mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all cell lines tested independently of the status of Deptor, PTEN, and PI3K. All cell lines expressed either Raptor, Rictor or both; excepting H929 and U266LR7 which were negative for both of them. Moreover, primary plasma cells from several MM patients highly expressed pS6R while normal cells were negative for this protein. We found that INK128 and rapamycin effectively suppressed phosphorylation of p6SR, but only INK128 was able to decrease phosphorylation of 4E-BP1. We observed that INK128 fully suppressed cell viability in a dose and time dependent manner, but rapamycin reached a plateau in efficacy at ± 60%. The IC50 of INK128 was in the range of 7.5–30 nM in the eight cell lines tested. Similar results were observed in freshly isolated plasma cells from MM patients. Besides the induction of apoptosis and cell cycle arrest, INK128 was more potent than rapamycin to induce autophagy, and only INK128 was able to induce PARP and Caspases 3, 8 and 9 cleavage. In the bone marrow microenvironment context, INK128 inhibited the proliferation of MM cells and decreased the p4E-BP1 induction. Importantly, treatment with rapamycin under such conditions did not affect cell proliferation. INK128 also showed a significantly greater effect inhibiting cell adhesion to fibronectin OPM2 MM1S, BMSCs and HUVECs compared to rapamycin. These results were confirmed in vivo. Oral daily treatment of NK128 (1.0 mg/kg) decreased tumor growth and improved survival of mice implanted with MM1S. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

The purpose of this study was to evaluate the anti-leukemic and immunosuppressive properties of novel, ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR). The mTOR inhibitors currently in clinical use, exemplified by rapamycin and related analogs (“rapalogs”), have achieved some success as anticancer therapies. However, these agents also suppress proliferation of both B and T lymphocytes. In addition, rapalogs have a mechanistic limitation: not only are they allosteric inhibitors that selectively bind to mTOR complex-1 (TORC1) and not to mTOR complex-2 (TORC2), but they do not fully inhibit TORC1. In contrast, novel ATP-competitive active-site inhibitors of the mTOR kinase overcome these limitations, displaying full inhibitory activity of both TORC1 and TORC2. We compared rapamycin with active-site TORC1/2 inhibitors in models of Philadelphia chromosome-positive pre-B acute lymphoblastic leukemia (Ph+ B-ALL). We find that PP242 and INK128, active-site TORC1/2 inhibitors, but not rapamycin cause death of murine and primary human leukemia cells. These compounds are also more potent than rapamycin in growth assays of solid tumor cell lines. Biochemical assays confirm that PP242 and INK128 inhibit rapamycin-resistant outputs of both TORC1 and TORC2. In vivo, oral administration of PP242 or INK128 delays leukemia onset and augments the effects of tyrosine kinase inhibitors. Surprisingly, active-site TORC1/2 inhibitors have much weaker effects on proliferation and function of normal B and T cells than rapamycin. Selective TORC1/2 inhibitors are also less immunosuppressive than PI-103, a panPI3K-mTORC1/2 inhibitor. These findings establish that transformed lymphocytes are selectively sensitive to TORC1/2 inhibitors and support the development of such inhibitors for leukemia therapy. 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 1798.

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.

Anti-angiogenic agents have demonstrated promising activity in patients with renal cell carcinoma (RCC). Patients with clear cell RCC often have mutations or silencing of the von Hippel-Lindau gene, leading to an accumulation of HIF 1 alpha (HIF1α), a key mediator of hypoxia-triggered neo-angiogenesis. The mammalian target of rapamycin (mTOR) is upstream of HIF1a and downstream of the VEGF pathway. mTOR kinase comprises two distinct multi-protein complexes, TORC1 and TORC2, which together regulate growth, metabolism, angiogenesis and survival. Pharmaceutical derivatives of rapamycin, a partial allosteric inhibitor of TORC1, provide clinical proof of concept for targeting mTOR in RCC as well as insights into how ATP-competitive TORC1/2 inhibitors might provide superior efficacy. Through rational drug design we have discovered INK128, a potent, selective TORC1/2 inhibitor with outstanding drug-like properties. We investigated INK128 in preclinical in vitro and in vivo models of RCC. Interestingly, while both INK128 and rapamycin exhibit comparable anti-angiogenic activity in vitro, INK128 correlated with potent and complete blockade of cell proliferation, while rapamycin failed to establish a dose-dependent maximum inhibition of tumor cell proliferation. A comparison of INK128, rapamycin, Nexavar and Avastin demonstrated that all exhibited potent inhibition of tumor growth, via different molecular mechanisms. INK128 inhibits phosphorylation of AKT, S6 and 4EBP1; rapamycin inhibits only S6 phosphorylation and induces AKT phosphorylation; Nexavar and Avastin have little effect on the PI3K/AKT/mTOR pathway. Only INK128 induced autophagy and decreased expression of cyclin D1. INK128 and rapamycin both inhibit expression of HIF-1α and VEGF, which contributes to their anti-angiogenic activity. We conclude that, although Nexavar, Avastin and rapamycin exert their activity primarily through effects on the tumor microenvironment, the anti-tumor activity of INK128 is derived from direct inhibition of tumor cell growth as well as anti-angiogenic activities. In summary, targeting TORC1/2 signaling with INK128 offers a compelling approach to the treatment of RCC. 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 4488.

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.