Tipifarnib as a Precision Therapy for HRAS-Mutant Head and Neck Squamous Cell Carcinomas.

Tipifarnib is a potent and highly selective inhibitor of farnesyltransferase (FTase). FTase catalyzes the posttranslational attachment of farnesyl groups to signaling proteins that are required for localization to cell membranes. Although all RAS isoforms are FTase substrates, only HRAS is exclusively dependent upon farnesylation, raising the possibility that HRAS-mutant tumors might be susceptible to tipifarnib-mediated inhibition of FTase. Here, we report the characterization of tipifarnib activity in a wide panel of HRAS-mutant and wild-type head and neck squamous cell carcinoma (HNSCC) xenograft models. Tipifarnib treatment displaced both mutant and wild-type HRAS from membranes but only inhibited proliferation, survival, and spheroid formation of HRAS-mutant cells. In vivo, tipifarnib treatment induced tumor stasis or regression in all six HRAS-mutant xenografts tested but displayed no activity in six HRAS wild-type patient-derived xenograft (PDX) models. Mechanistically, drug treatment resulted in the reduction of MAPK pathway signaling, inhibition of proliferation, induction of apoptosis, and robust abrogation of neovascularization, apparently via effects on both tumor cells and endothelial cells. Bioinformatics and quantitative image analysis further revealed that FTase inhibition induces progressive squamous cell differentiation in tipifarnib-treated HNSCC PDXs. These preclinical findings support that HRAS represents a druggable oncogene in HNSCC through FTase inhibition by tipifarnib, thereby identifying a precision therapeutic option for HNSCCs harboring HRAS mutations.

Development of combination therapies to maximize the impact of KRAS-G12C inhibitors in lung cancer.

KRAS represents an excellent therapeutic target in lung cancer, the most commonly mutated form of which can now be blocked using KRAS-G12C mutant-specific inhibitory trial drugs. Lung adenocarcinoma cells harboring KRAS mutations have been shown previously to be selectively sensitive to inhibition of mitogen-activated protein kinase kinase (MEK) and insulin-like growth factor 1 receptor (IGF1R) signaling. Here, we show that this effect is markedly enhanced by simultaneous inhibition of mammalian target of rapamycin (mTOR) while maintaining selectivity for the KRAS-mutant genotype. Combined mTOR, IGF1R, and MEK inhibition inhibits the principal signaling pathways required for the survival of KRAS-mutant cells and produces marked tumor regression in three different KRAS-driven lung cancer mouse models. Replacing the MEK inhibitor with the mutant-specific KRAS-G12C inhibitor ARS-1620 in these combinations is associated with greater efficacy, specificity, and tolerability. Adding mTOR and IGF1R inhibitors to ARS-1620 greatly improves its effectiveness on KRAS-G12C mutant lung cancer cells in vitro and in mouse models. This provides a rationale for the design of combination treatments to enhance the impact of the KRAS-G12C inhibitors, which are now entering clinical trials.

KRAS G12C NSCLC Models Are Sensitive to Direct Targeting of KRAS in Combination with PI3K Inhibition.

PURPOSE: KRAS-mutant lung cancers have been recalcitrant to treatments including those targeting the MAPK pathway. Covalent inhibitors of KRAS p.G12C allele allow for direct and specific inhibition of mutant KRAS in cancer cells. However, as for other targeted therapies, the therapeutic potential of these inhibitors can be impaired by intrinsic resistance mechanisms. Therefore, combination strategies are likely needed to improve efficacy.Experimental Design: To identify strategies to maximally leverage direct KRAS inhibition we defined the response of a panel of NSCLC models bearing the KRAS G12C-activating mutation in vitro and in vivo. We used a second-generation KRAS G12C inhibitor, ARS1620 with improved bioavailability over the first generation. We analyzed KRAS downstream effectors signaling to identify mechanisms underlying differential response. To identify candidate combination strategies, we performed a high-throughput drug screening across 112 drugs in combination with ARS1620. We validated the top hits in vitro and in vivo including patient-derived xenograft models. RESULTS: Response to direct KRAS G12C inhibition was heterogeneous across models. Adaptive resistance mechanisms involving reactivation of MAPK pathway and failure to induce PI3K-AKT pathway inactivation were identified as likely resistance events. We identified several model-specific effective combinations as well as a broad-sensitizing effect of PI3K-AKT-mTOR pathway inhibitors. The G12Ci+PI3Ki combination was effective in vitro and in vivo on models resistant to single-agent ARS1620 including patient-derived xenografts models. CONCLUSIONS: Our findings suggest that signaling adaptation can in some instances limit the efficacy of ARS1620 but combination with PI3K inhibitors can overcome this resistance.

An Internally Controlled Quantitative Target Occupancy Assay for Covalent Inhibitors.

Assessing target occupancy is critical for establishing proof-of-mechanism for novel inhibitors and to determine whether robust target inhibition can be achieved at tolerated doses. This is challenging in the clinic using conventional methods due to the need for untreated controls. We describe a new mass spectrometry approach to quantitatively assess target occupancy for covalent inhibitors that does not require untreated controls, and apply the method to the KRASG12C inhibitor ARS-1620.

The reactivity-driven biochemical mechanism of covalent KRASG12C inhibitors.

Activating mutations in KRAS are among the most common tumor driver mutations. Until recently, KRAS had been considered undruggable with small molecules; the discovery of the covalent KRASG12C inhibitors ARS-853 and ARS-1620 has demonstrated that it is feasible to inhibit KRAS with high potency in cells and animals. Although the biological activity of these inhibitors has been described, the biochemical mechanism of how the compounds achieve potent inhibition remained incompletely understood. We now show that the activity of ARS-853 and ARS-1620 is primarily driven by KRAS-mediated catalysis of the chemical reaction with Cys12 in human KRASG12C, while the reversible binding affinity is weak, in the hundreds of micromolar or higher range. The mechanism resolves how an induced, shallow and dynamic pocket not expected to support high-affinity binding of small molecules can nevertheless be targeted with potent inhibitors and may be applicable to other targets conventionally considered undruggable.

Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor.

KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.

Selective Inhibition of Oncogenic KRAS Output with Small Molecules Targeting the Inactive State.

UNLABELLED: KRAS gain-of-function mutations occur in approximately 30% of all human cancers. Despite more than 30 years of KRAS-focused research and development efforts, no targeted therapy has been discovered for cancers with KRAS mutations. Here, we describe ARS-853, a selective, covalent inhibitor of KRAS(G12C) that inhibits mutant KRAS-driven signaling by binding to the GDP-bound oncoprotein and preventing activation. Based on the rates of engagement and inhibition observed for ARS-853, along with a mutant-specific mass spectrometry-based assay for assessing KRAS activation status, we show that the nucleotide state of KRAS(G12C) is in a state of dynamic flux that can be modulated by upstream signaling factors. These studies provide convincing evidence that the KRAS(G12C) mutation generates a "hyperexcitable" rather than a "statically active" state and that targeting the inactive, GDP-bound form is a promising approach for generating novel anti-RAS therapeutics. SIGNIFICANCE: A cell-active, mutant-specific, covalent inhibitor of KRAS(G12C) is described that targets the GDP-bound, inactive state and prevents subsequent activation. Using this novel compound, we demonstrate that KRAS(G12C) oncoprotein rapidly cycles bound nucleotide and responds to upstream signaling inputs to maintain a highly active state.

Resistance to mTOR kinase inhibitors in lymphoma cells lacking 4EBP1.

Inhibitors of the mechanistic target of rapamycin (mTOR) hold promise for treatment of hematological malignancies. Analogs of the allosteric mTOR inhibitor rapamycin are approved for mantle cell lymphoma but have limited efficacy in other blood cancers. ATP-competitive "active-site" mTOR inhibitors produce more complete mTOR inhibition and are more effective than rapamycin in preclinical models of leukemia, lymphoma and multiple myeloma. In parallel to clinical trials of active-site mTOR inhibitors, it will be important to identify resistance mechanisms that might limit drug efficacy in certain patients. From a panel of diffuse large B-cell lymphoma cell lines, we found that the VAL cell line is particularly resistant to apoptosis in the presence of active-site mTOR inhibitors. Mechanistic investigation showed that VAL does not express eukaryotic initiation factor 4E-binding protein-1 (4EBP1), a key negative regulator of translation controlled by mTOR. Although VAL cells express the related protein 4EBP2, mTOR inhibitor treatment fails to displace eukaryotic initiation factor 4G from the mRNA cap-binding complex. Knockdown of eukaryotic initiation factor 4E, or re-expression of 4EBP1, sensitizes cells to apoptosis when treated with active-site mTOR inhibitors. These findings provide a naturally occurring example of 4EBP deficiency driving lymphoma cell resistance to active-site mTOR inhibitors.

Targeting of the MNK-eIF4E axis in blast crisis chronic myeloid leukemia inhibits leukemia stem cell function.

Chronic myeloid leukemia responds well to therapy targeting the oncogenic fusion protein BCR-ABL1 in chronic phase, but is resistant to treatment after it progresses to blast crisis (BC). BC is characterized by elevated ß-catenin signaling in granulocyte macrophage progenitors (GMPs), which enables this population to function as leukemia stem cells (LSCs) and act as a reservoir for resistance. Because normal hematopoietic stem cells (HSCs) and LSCs depend on ß-catenin signaling for self-renewal, strategies to specifically target BC will require identification of drugable factors capable of distinguishing between self-renewal in BC LSCs and normal HSCs. Here, we show that the MAP kinase interacting serine/threonine kinase (MNK)-eukaryotic translation initiation factor 4E (eIF4E) axis is overexpressed in BC GMPs but not normal HSCs, and that MNK kinase-dependent eIF4E phosphorylation at serine 209 activates ß-catenin signaling in BC GMPs. Mechanistically, eIF4E overexpression and phosphorylation leads to increased ß-catenin protein synthesis, whereas MNK-dependent eIF4E phosphorylation is required for nuclear translocation and activation of ß-catenin. Accordingly, we found that a panel of small molecule MNK kinase inhibitors prevented eIF4E phosphorylation, ß-catenin activation, and BC LSC function in vitro and in vivo. Our findings identify the MNK-eIF4E axis as a specific and critical regulator of BC self-renewal, and suggest that pharmacologic inhibition of the MNK kinases may be therapeutically useful in BC chronic myeloid leukemia.

Selective inhibition of phosphoinositide 3-kinase p110α preserves lymphocyte function.

Class IA phosphoinositide 3-kinase (PI3K) is essential for clonal expansion, differentiation, and effector function of B and T lymphocytes. The p110δ catalytic isoform of PI3K is highly expressed in lymphocytes and plays a prominent role in B and T cell responses. Another class IA PI3K catalytic isoform, p110α, is a promising drug target in cancer but little is known about its function in lymphocytes. Here we used highly selective inhibitors to probe the function of p110α in lymphocyte responses in vitro and in vivo. p110α inhibition partially reduced B cell receptor (BCR)-dependent AKT activation and proliferation, and diminished survival supported by the cytokines BAFF and IL-4. Selective p110δ inhibition suppressed B cell responses much more strongly, yet maximal suppression was achieved by targeting multiple PI3K isoforms. In mouse and human T cells, inhibition of single class IA isoforms had little effect on proliferation, whereas pan-class I inhibition did suppress T cell expansion. In mice, selective p110α inhibition using the investigational agent MLN1117 (previously known as INK1117) did not disrupt the marginal zone B cell compartment and did not block T cell-dependent germinal center formation. In contrast, the selective p110δ inhibitor IC87114 strongly suppressed germinal center formation and reduced marginal zone B cell numbers, similar to a pan-class I inhibitor. These findings show that although acute p110α inhibition partially diminishes AKT activation, selective p110α inhibitors are likely to be less immunosuppressive in vivo compared with p110δ or pan-class I inhibitors.

The translational landscape of mTOR signalling steers cancer initiation and metastasis.

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.

The in vivo evaluation of active-site TOR inhibitors in models of BCR-ABL+ leukemia.

Preclinical evaluation of candidate anticancer compounds requires appropriate animal models. Most commonly, solid tumor xenograft systems are employed in which immunocompromised mice are implanted with human cancer cell lines. Genetically engineered mouse models of solid tumors are also frequently employed. Both of these approaches can also be applied to studies of hematological malignancies. In this chapter, we describe three types of mouse models of leukemia driven by the human BCR-ABL oncogene. We also discuss the application of these models to preclinical testing of active-site TOR inhibitors, a novel class of compounds that selectively target the ATP-binding pocket of the target of rapamycin (TOR) kinase.

Sphingolipid-based drugs selectively kill cancer cells by down-regulating nutrient transporter proteins.

Cancer cells are hypersensitive to nutrient limitation because oncogenes constitutively drive glycolytic and TCA (tricarboxylic acid) cycle intermediates into biosynthetic pathways. As the anaplerotic reactions that replace these intermediates are fueled by imported nutrients, the cancer cell's ability to generate ATP becomes compromised under nutrient-limiting conditions. In addition, most cancer cells have defects in autophagy, the catabolic process that provides nutrients from internal sources when external nutrients are unavailable. Normal cells, in contrast, can adapt to the nutrient stress that kills cancer cells by becoming quiescent and catabolic. In the present study we show that FTY720, a water-soluble sphingolipid drug that is effective in many animal cancer models, selectively starves cancer cells to death by down-regulating nutrient transporter proteins. Consistent with a bioenergetic mechanism of action, FTY720 induced homoeostatic autophagy. Cells were protected from FTY720 by cell-permeant nutrients or by reducing nutrient demand, but blocking apoptosis was ineffective. Importantly, AAL-149, a FTY720 analogue that lacks FTY720's dose-limiting toxicity, also triggered transporter loss and killed patient-derived leukaemias while sparing cells isolated from normal donors. As they target the metabolic profile of cancer cells rather than specific oncogenic mutations, FTY720 analogues such as AAL-149 should be effective against many different tumour types, particularly in combination with drugs that inhibit autophagy.

B-1 cell lymphoma in mice lacking the steroid and xenobiotic receptor, SXR.

The steroid and xenobiotic receptor (SXR) is a broad-specificity nuclear hormone receptor that is highly expressed in the liver and intestine, where its primary function is to regulate drug and xenobiotic metabolism. SXR is expressed at lower levels in other tissues, where little is known about its physiological functions. We previously linked SXR with immunity and inflammation by showing that SXR antagonizes the activity of nuclear factor (NF)-κB in vitro and in vivo. SXR(-/-) mice demonstrate aberrantly high NF-κB activity and overexpression of NF-κB target genes. Here we show that SXR(-/-) mice develop B cell lymphoma in an age-dependent manner. SXR(-/-) mice develop multiple hyperplastic lymphoid foci composed of B-1a cells in the intestine, spleen, lymph nodes, peritoneal cavity, and blood. In all circumstances, these lymphocytes possess cell surface and molecular characteristics of either chronic lymphocytic leukemia or non-Hodgkin's lymphoma originating from B-1 lymphocytes. These results demonstrate a novel and unsuspected role for SXR signaling in the B-1 cell compartment, establish SXR as a tumor suppressor in B-1 cells, and may provide a link between metabolism of xenobiotic compounds and lymphomagenesis.

Targeting TOR dependence in cancer.

A challenge in cancer therapy has been to identify targets whose function is essential for survival of malignant cells but not normal cells. This Perspective discusses recent evidence that novel inhibitors of the kinase TOR can provide an unprecedented balance of anti-cancer efficacy and tolerability.

Effective and selective targeting of leukemia cells using a TORC1/2 kinase inhibitor.

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.

Immune regulation by rapamycin: moving beyond T cells.

The mammalian target of rapamycin (mTOR) is a multifunctional kinase that promotes cell growth and division in response to growth factor and nutrient signals. Rapamycin exerts its potent immunosuppressive effects in part through direct effects on antigen-specific lymphocytes; however, rapamycin also modulates adaptive immunity through its effects on innate immune cells, including dendritic cells and macrophages. Studies have established rapamycin-sensitive functions of mTOR, downstream of Toll-like receptors, in shaping the cytokine response of myeloid cells and driving the production of interferon by plasmacytoid dendritic cells. These findings point to new strategies for boosting or suppressing specific immune responses.

Ablation of PI3K blocks BCR-ABL leukemogenesis in mice, and a dual PI3K/mTOR inhibitor prevents expansion of human BCR-ABL+ leukemia cells.

Some cases of pre-B cell acute lymphoblastic leukemia (pre-B-ALL) are caused by the Philadelphia (Ph) chromosome-encoded BCR-ABL oncogene, and these tend to have a poor prognosis. Inhibitors of the PI3K/AKT pathway reduce BCR-ABL-mediated transformation in vitro; however, the specific PI3K isoforms involved are poorly defined. Using a murine model of Ph+ pre-B-ALL, we found that deletion of both Pik3r1 and Pik3r2, genes encoding class IA PI3K regulatory isoforms, severely impaired transformation. BCR-ABL-dependent pre/pro-B cell lines could be established at low frequency from progenitors that lacked these genes, but the cells were smaller, proliferated more slowly, and failed to cause leukemia in vivo. These cell lines displayed nearly undetectable PI3K signaling function and were resistant to the PI3K inhibitor wortmannin. However, they maintained activation of mammalian target of rapamycin (mTOR) and were more sensitive to rapamycin. Treatment with rapamycin caused feedback activation of AKT in WT cell lines but not PI3K-deficient lines. A dual inhibitor of PI3K and mTOR, PI-103, was more effective than rapamycin at suppressing proliferation of mouse pre-B-ALL and human CD19+CD34+)Ph+ ALL leukemia cells treated with the ABL kinase inhibitor imatinib. Our findings provide mechanistic insights into PI3K dependency in oncogenic networks and provide a rationale for targeting class IA PI3K, alone or together with mTOR, in the treatment of Ph+ ALL.