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Supplementary Figure 1

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.

Supplementary Table 1

Supplementary Data 3

Legends to supplementary table, data and figures

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.

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.

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 KRAS

We tested the interaction of 72 kinase inhibitors with 442 kinases covering >80% of the human catalytic protein kinome. Our data show that, as a class, type II inhibitors are more selective than type I inhibitors, but that there are important exceptions to this trend. The data further illustrate that selective inhibitors have been developed against the majority of kinases targeted by the compounds tested. Analysis of the interaction patterns reveals a class of 'group-selective' inhibitors broadly active against a single subfamily of kinases, but selective outside that subfamily. The data set suggests compounds to use as tools to study kinases for which no dedicated inhibitors exist. It also provides a foundation for further exploring kinase inhibitor biology and toxicity, as well as for studying the structural basis of the observed interaction patterns. Our findings will help to realize the direct enabling potential of genomics for drug development and basic research about cellular signaling.

SummaryInteractions between kinases and small molecule inhibitors can be activation state dependent. A detailed understanding of inhibitor binding therefore requires characterizing interactions across multiple activation states. We have systematically explored the effects of ABL1 activation loop phosphorylation and PDGFR family autoinhibitory juxtamembrane domain docking on inhibitor binding affinity. For a diverse compound set, the affinity patterns correctly classify inhibitors as having type I or type II binding modes, and we show that juxtamembrane domain docking can have dramatic negative effects on inhibitor affinity. The results have allowed us to associate ligand-induced conformational changes observed in cocrystal structures with specific energetic costs. The approach we describe enables investigation of the complex relationship between kinase activation state and compound binding affinity and should facilitate strategic inhibitor design.

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.

Patients with MLL-rearranged leukemia typically have a poor prognosis and no targeted therapies are yet available. As the leukemogenic activity of MLL fusion proteins has been shown to be dependent on their direct interaction with menin, development of small molecules that block the menin-MLL interaction is a promising therapeutic strategy for this disease. In addition, recent reports have implicated menin-MLL signaling in some forms of MLL wild-type AML, including those bearing oncogenic mutations in NPM1 and DNMT3A, which together represent 45% of cases of AML, suggesting that menin-MLL inhibitors could provide clinical benefit in a broader population of AML patients than originally envisioned. We have recently reported a novel, potent, and selective small molecule menin-MLL inhibitor, KO-539, that effectively treats MLL leukemias in in vivo models, demonstrating its potential clinical utility. The compound potently inhibits the growth of MLL-rearranged cell lines, displays favorable PK properties, and is remarkably effective in a subcutaneous MV4;11 xenograft model. In both the MV4;11 and MOLM13 disseminated leukemia models, the compound confers a prolonged survival benefit compared to vehicle or the Phase III-stage FLT3 inhibitor quizartinib when dosed orally daily at well-tolerated doses. To extend the characterization of the compound to additional mutationally-defined subsets of AML, the activity of KO-539 was compared to quizartinib in two disseminated patient-derived xenograft (PDX) models of AML. In two independent studies in an NPM1mut/DNMT3Amut/FLT3-ITD model, animals received daily oral treatment KO-539, and tumor progression was monitored by weekly sampling and multiparameter FACS analysis of circulating human blasts, with similar analysis of bone marrow and spleen at sacrifice. All vehicle treated animals displayed progressive leukemia and died between 15 and 25 days after initiation of therapy with bone marrow completely comprised of undifferentiated CD45/CD38+ blasts and grossly enlarged spleens. By contrast, all animals treated with KO-539 were cleared of detectable leukemia by Day 28 and, in the 40% surviving animals in the first study, leukemia was not detectable even four weeks after cessation of therapy. Interestingly, those KO-539-treated animals that did succumb were found to have normal spleen weight and bone marrow that contained only differentiated CD11b+ human monocytes, suggesting that the compound was pharmacologically effective in all animals. This finding was confirmed in the repeat study, where therapy was initiated a few days earlier and all animals had no detectable tumors two months later. By contrast, quizartinib-treated animals initially responded well but relapsed while on treatment. Importantly, these results were confirmed in a second NPM1mut/DNMT3A-WT/FLT3 mut PDX model, where vehicle-treated animals succumbed by Day 35 and quizartinib-treated animals relapsed with progressive leukemia around the same time, but no evidence of disease progression was evident in the KO-539 group after 56 days. We describe a menin-MLL inhibitor with optimized drug-like properties that demonstrates potential clinical utility in preclinical models of MLL-rearranged and NPM1/DNMT3A/FLT3-mutant acute myeloid leukemias. The compound is currently undergoing further preclinical evaluation. Citation Format: Francis Burrows, Tao Wu, Linda Kessler, Shuangwei Li, Jingchuan Zhang, Patrick Zarrinkar, Liansheng Li, Tomasz Cierpicki, Jolanta Grembecka, Pingda Ren, Yi Liu. A novel small molecule menin-MLL inhibitor for potential treatment of MLL-rearranged leukemias and NPM1/DNMT3A-mutant AML [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A27.

Activating mutations in the receptor tyrosine kinase FLT3 are present in up to approximately 30% of acute myeloid leukemia (AML) patients, implicating FLT3 as a driver of the disease and therefore as a target for therapy. We report the characterization of AC220, a second-generation FLT3 inhibitor, and a comparison of AC220 with the first-generation FLT3 inhibitors CEP-701, MLN-518, PKC-412, sorafenib, and sunitinib. AC220 exhibits low nanomolar potency in biochemical and cellular assays and exceptional kinase selectivity, and in animal models is efficacious at doses as low as 1 mg/kg given orally once daily. The data reveal that the combination of excellent potency, selectivity, and pharmacokinetic properties is unique to AC220, which therefore is the first drug candidate with a profile that matches the characteristics desirable for a clinical FLT3 inhibitor.

Treatment of AML patients with small molecule inhibitors of FLT3 kinase has been explored as a viable therapy. However, these agents are found to be less than optimal for the treatment of AML because of lack of sufficient potency or suboptimal oral pharmacokinetics (PK) or lack of adequate tolerability at efficacious doses. We have developed a series of extremely potent and highly selective FLT3 inhibitors with good oral PK properties. The first series of compounds represented by 1 (AB530) was found to be a potent and selective FLT3 kinase inhibitor with good PK properties. The aqueous solubility and oral PK properties at higher doses in rodents were found to be less than optimal for clinical development. A novel series of compounds were designed lacking the carboxamide group of 1 with an added water solubilizing group. Compound 7 (AC220) was identified from this series to be the most potent and selective FLT3 inhibitor with good pharmaceutical properties, excellent PK profile, and superior efficacy and t...

To fully exploit the potential of kinases as drug targets, novel strategies for the efficient discovery of inhibitors are required. In contrast to the traditional, linear process of inhibitor discovery, high-throughput kinase profiling enables a parallel approach by interrogating compounds against hundreds of targets in a single screen. Compound potency and selectivity are determined simultaneously, providing a choice of targets to pursue that is guided by the quality of lead compounds available, rather than by target biology alone.

Kinase inhibitors are a new class of therapeutics with a propensity to inhibit multiple targets. The biological consequences of multi-kinase activity are poorly defined, and an important step toward understanding the relationship between selectivity, efficacy and safety is the exploration of how inhibitors interact with the human kinome. We present interaction maps for 38 kinase inhibitors across a panel of 317 kinases representing >50% of the predicted human protein kinome. The data constitute the most comprehensive study of kinase inhibitor selectivity to date and reveal a wide diversity of interaction patterns. To enable a global analysis of the results, we introduce the concept of a selectivity score as a general tool to quantify and differentiate the observed interaction patterns. We further investigate the impact of panel size and find that small assay panels do not provide a robust measure of selectivity.

Based on a putative binding mode of quizartinib (AC220, 1), a potent FMS-like tyrosine kinase 3 (FLT3) inhibitor in Phase III clinical development, we have designed de novo a simpler aminopyridine-based hinge binding motif. Further optimization focusing on maximizing in vivo efficacy and minimizing CYP3A4 time-dependent inhibition resulted in a highly efficacious compound (6s) in tumor xenograft model for further preclinical development.

Patients with MLL-rearranged leukemia typically have a poor prognosis. With chemotherapy and stem cell transplantation as current standard of care, the 5-year survival rate is estimated to be only about 35%. As the leukemogenic activity of MLL fusion proteins has been shown to be dependent on their direct interaction with menin, development of small molecules that block the menin-MLL interaction is a promising therapeutic strategy for the treatment of this disease. Although small molecule menin-MLL inhibitors have been reported, previously published compounds exhibited modest cellular potency and/or poor pharmacokinetic properties. We now describe a novel, potent, and selective small molecule menin-MLL inhibitor that effectively treats MLL leukemias in in vivo models and demonstrate its potential clinical utility. The compound selectively inhibits the growth of a panel of MLL-rearranged cell lines relative to non MLL-rearranged cell lines, displays favorable pharmacokinetic properties, and induces tumor regression in an MV4;11 subcutaneous xenograft mouse model, with durable tumor suppression even after dosing is discontinued. In both the MV4;11 and MOLM13 disseminated leukemia models, the compound confers a prolonged survival benefit compared to an untreated group when dosed orally once daily at doses that are well-tolerated in the animals. We have used this potent and selective menin inhibitor to investigate the functional cellular response of MLL-rearranged cell lines to menin inhibition, assessing cell growth, cell cycle and gene expression responses over time, and compared that response to other epigenetic inhibitors and demonstrate significantly increased apoptosis which correlates with tumor regression measured both in early and long term readouts in xenograft and systemic leukemia models. We describe a menin-MLL inhibitor with optimized drug-like properties that demonstrates potential clinical utility in preclinical models of MLL leukemias. The compound is currently under further preclinical evaluation. Citation Format: Tao Wu, Linda Kessler, Shuangwei Li, Trupta Purohit, Shisheng Li, Hongzhi Miao, Brian Linhares, Rasmus Hansen, Jeff Kucharski, Yi Wang, Ke Yu, Katarzyna Kempinska, Tess Ely, Szymon Klossowski, Ata Zarieh, Ulf Peters, Jun Feng, Yvonne Yao, Yuan Liu, Bo Wen, Francis Burrows, Duxin Sun, Jingchuan Zhang, Levan Darjania, Dana Hu-Lowe, Patrick Zarrinkar, Liansheng Li, Tomasz Cierpicki, Jolanta Grembecka, Pingda Ren, Yi Liu. A novel small molecule menin-MLL inhibitor for potential treatment of MLL-rearranged leukemias [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5077. doi:10.1158/1538-7445.AM2017-5077

Epithelial ovarian cancer is the leading cause of death from gynecologic cancer, in part because of the lack of effective early detection methods. Although alterations of several genes, such as c-erb-B2, c-myc , and p53 , have been identified in a significant fraction of ovarian cancers, none of these mutations are diagnostic of malignancy or predictive of tumor behavior over time. Here, we used oligonucleotide microarrays with probe sets complementary to >6,000 human genes to identify genes whose expression correlated with epithelial ovarian cancer. We extended current microarray technology by simultaneously hybridizing ovarian RNA samples in a highly parallel manner to a single glass wafer containing 49 individual oligonucleotide arrays separated by gaskets within a custom-built chamber (termed “array-of-arrays”). Hierarchical clustering of the expression data revealed distinct groups of samples. Normal tissues were readily distinguished from tumor tissues, and tumors could be further subdivided into major groupings that correlated both to histological and clinical observations, as well as cell type-specific gene expression. A metric was devised to identify genes whose expression could be considered ideal for molecular determination of epithelial ovarian malignancies. The list of genes generated by this method was highly enriched for known markers of several epithelial malignancies, including ovarian cancer. This study demonstrates the rapidity with which large amounts of expression data can be generated. The results highlight important molecular features of human ovarian cancer and identify new genes as candidate molecular markers.

SummarySelective protein kinase inhibitors have only been developed against a small number of kinase targets. Here we demonstrate that “high-throughput kinase profiling” is an efficient method for the discovery of lead compounds for established as well as unexplored kinase targets. We screened a library of 118 compounds constituting two distinct scaffolds (furan-thiazolidinediones and pyrimido-diazepines) against a panel of 353 kinases. A distinct kinase selectivity profile was observed for each scaffold. Selective inhibitors were identified with submicromolar cellular activity against PIM1, ERK5, ACK1, MPS1, PLK1-3, and Aurora A,B kinases. In addition, we identified potent inhibitors for so far unexplored kinases such as DRAK1, HIPK2, and DCAMKL1 that await further evaluation. This inhibitor-centric approach permits comprehensive assessment of a scaffold of interest and represents an efficient and general strategy for identifying new selective kinase inhibitors.

Treatment of AML patients with small molecule inhibitors of FLT3 kinase has been explored as a viable therapy. However, these agents are found to be less than optimal for the treatment of AML because of lack of sufficient potency or suboptimal oral pharmacokinetics (PK) or lack of adequate tolerability at efficacious doses. We have developed a series of extremely potent and highly selective FLT3 inhibitors with good oral PK properties. The first series of compounds represented by 1 (AB530) was found to be a potent and selective FLT3 kinase inhibitor with good PK properties. The aqueous solubility and oral PK properties at higher doses in rodents were found to be less than optimal for clinical development. A novel series of compounds were designed lacking the carboxamide group of 1 with an added water solubilizing group. Compound 7 (AC220) was identified from this series to be the most potent and selective FLT3 inhibitor with good pharmaceutical properties, excellent PK profile, and superior efficacy and tolerability in tumor xenograft models. Compound 7 has demonstrated a desirable safety and PK profile in humans and is currently in phase II clinical trials.

A series of diaryl ureas with an amide substitution at the 4-position was prepared and found to be potent and selective FLT3 inhibitors with good oral bioavailability and efficacy in a tumor xenograft model.

The upstream protein kinases responsible for thousands of phosphorylation events in the phosphoproteome remain to be discovered. We developed a three-component chemical reaction which converts the transient noncovalent substrate-kinase complex into a covalently cross-linked product by utilizing a dialdehyde-based cross-linker, 1. Unfortunately, the reaction of 1 with a lysine in the kinase active site and an engineered cysteine on the substrate to form an isoindole cross-linked product could not be performed in the presence of competing cellular proteins due to nonspecific side reactions. In order to more selectively target the cross-linker to protein kinases in cell lysates, we replaced the weak, kinase-binding adenosine moiety of 1 with a potent protein kinase inhibitor scaffold. In addition, we replaced the o-phthaldialdehyde moiety in 1 with a less-reactive thiophene-2,3-dicarboxaldehyde moiety. The combination of these two structural modifications provides for cross-linking of a cysteine-containing substrate to its corresponding kinase in the presence of competing cellular proteins.

We present a high-resolution (2.0 Å) crystal structure of the catalytic domain of a mutant form of the Abl tyrosine kinase (H396P; Abl-1a numbering) that is resistant to the Abl inhibitor imatinib. The structure is determined in complex with the small-molecule inhibitor VX-680 (Vertex Pharmaceuticals, Cambridge, MA), which blocks the activity of various imatinib-resistant mutant forms of Abl, including one (T315I) that is resistant to both imatinib and BMS-354825 (dasatinib), a dual Src/Abl inhibitor that seems to be clinically effective against all other imatinib-resistant forms of BCR-Abl. VX-680 is shown to have significant inhibitory activity against BCR-Abl bearing the T315I mutation in patient-derived samples. The Abl kinase domain bound to VX-680 is not phosphorylated on the activation loop in the crystal structure but is nevertheless in an active conformation, previously unobserved for Abl and inconsistent with the binding of imatinib. The adoption of an active conformation is most likely the result of synergy between the His396Pro mutation, which destabilizes the inactive conformation required for imatinib binding, and the binding of VX-680, which favors the active conformation through hydrogen bonding and steric effects. VX-680 is bound to Abl in a mode that accommodates the substitution of isoleucine for threonine at residue 315 (the “gatekeeper” position). The avoidance of the innermost cavity of the Abl kinase domain by VX-680 and the specific recognition of the active conformation explain the effectiveness of this compound against mutant forms of BCR-Abl, including those with mutations at the gatekeeper position. (Cancer Res 2006; 66(2): 1007-14)

Kinase inhibitors show great promise as a new class of therapeutics. Here we describe an efficient way to determine kinase inhibitor specificity by measuring binding of small molecules to the ATP site of kinases. We have profiled 20 kinase inhibitors, including 16 that are approved drugs or in clinical development, against a panel of 119 protein kinases. We find that specificity varies widely and is not strongly correlated with chemical structure or the identity of the intended target. Many novel interactions were identified, including tight binding of the p38 inhibitor BIRB-796 to an imatinib-resistant variant of the ABL kinase, and binding of imatinib to the SRC-family kinase LCK. We also show that mutations in the epidermal growth factor receptor (EGFR) found in gefitinib-responsive patients do not affect the binding affinity of gefitinib or erlotinib. Our results represent a systematic small molecule-protein interaction map for clinical compounds across a large number of related proteins.

Gene expression profiling using DNA arrays is rapidly becoming an essential tool for research and drug discovery and may soon play a central role in disease diagnosis. Although it is possible to make significant discoveries on the basis of a relatively small number of expression profiles, the full potential of this technology is best realized through more extensive collections of expression measurements. The generation of large numbers of expression profiles can be a time-consuming and labor-intensive process with current one-at-a-time technology. We have developed the ability to obtain expression profiles in a highly parallel yet straightforward format using glass wafers that contain 49 individual high-density oligonucleotide arrays. This arrays of arrays concept is generalizable and can be adapted readily to other types of arrays, including spotted cDNA microarrays. It is also scalable for use with hundreds and even thousands of smaller arrays on a single piece of glass. Using the arrays of arrays approach and parallel preparation of hybridization samples in 96-well plates, we were able to determine the patterns of gene expression in 27 ovarian carcinomas and 4 normal ovarian tissue samples, along with a number of control samples, in a single experiment. This new approach significantly increases the ease, efficiency, and throughput of microarray-based experiments and makes possible new applications of expression profiling that are currently impractical.

Group I ribozymes can repair mutant RNAs via trans-splicing. Unfortunately, substrate specificity is quite low for the trans-splicing reaction catalyzed by the group I ribozyme from Tetrahymenathermophila. We have used a systematic approach based on biochemical knowledge of the function of the Tetrahymena ribozyme to optimize its ability to discriminate against nonspecific substrates in vitro. Ribozyme derivatives that combine a mutation which indirectly slows down the rate of the chemical cleavage step by weakening guanosine binding with additional mutations that weaken substrate binding have greatly enhanced specificity with short oligonucleotide substrates and an mRNA fragment derived from the p53 gene. Moreover, compared to the wild-type ribozyme, reaction of a more specific ribozyme with targeted substrates is much less sensitive to the presence of nonspecific RNA competitors. These results demonstrate how a detailed understanding of the biochemistry of a catalytic RNA can facilitate the design of customized ribozymes with improved properties for therapeutic applications.

One largely unexplored question about group I intron splicing is how the cleavage and ligation steps of the reaction are coordinated. We describe a simple in vitro trans-splicing model system in which both steps take place, including the exchange of ligands in the guanosine-binding site that must occur between the two steps. Using this model system, we show that the switch is accomplished by modulating the relative affinity of the binding site for the two ligands. While the terminal guanosine of the intron (omegaG) and exogenous guanosine compete for binding during the first step of splicing, no competition is apparent during the second step, when omegaG is bound tightly. These results help explain how the ribozyme orchestrates progression through the splicing reaction. In addition to providing a new tool to ask basic questions about RNA catalysis, the trans-splicing model system will also facilitate the development of therapeutically useful group I ribozymes that can repair mutant mRNAs.

Fms-like tyrosine kinase 3 (FLT3) is thought to be a major driver in the pathogenesis of acute myeloid leukemia (AML). FLT3-activating mutations are found in ∼ 30% of AML patients and are associated with poor outcome in this patient population. AC220 is a small molecule kinase inhibitor with potent and selective FLT3 inhibitory activity. Here we characterize the effect of AC220 on FLT3 autophosphorylation, activation of downstream signaling pathways (STAT5, ERK and AKT), cell cycle distribution, and apoptosis endpoints in multiple leukemia cell lines expressing either wild type FLT3 (FLT3-WT, SEM-K2 cells (FLT3-WT overexpression) and RS4;11 cells (FLT3-WT)) and/or ITD-mutated FLT3 (FLT3-ITD, MV4-11 (FLT3-ITD), MOLM-14 (FLT3-ITD/FLT3-WT)). Across each of these parameters we also compare the effects of AC220 to those of other known FLT3 inhibitors including, sorafenib, sunitinib, lestaurtinib, and midostaurin. AC220 inhibited FLT3 autophosphorylation in both FLT3-WT and FLT3-ITD cells with roughly equal potency, while other FLT3 inhibitors showed differential pFLT3 inhibition across several cell lines. Although AC220-mediated inhibition of FLT3 autophosphorylation was observed in all cell lines, subsequent cell death was only detected in the cell lines with activated FLT3. This contrasts with other FLT3 inhibitors, where potent inhibition of cell proliferation in the non-FLT3 activated RS4;11 cells was observed, highlighting the lack of FLT3 selectivity of these compounds. Consistent with FLT3 inhibition, AC220 also inhibited constitutively activated STAT5, ERK and AKT in these cell lines, suggesting that one or more of these pathways play a role in FLT3-mediated cell growth and survival. Furthermore, the inhibition of these downstream markers correlated with the inhibition of FLT3 across each of the FLT3 inhibitors tested. Lastly, in an MV4-11 murine xenograft model, AC220 administration resulted in the reduction of phosphorylated FLT3 and downstream signaling molecules concomitant with tumor reduction. Together, these data suggest that selective FLT3 inhibition is sufficient to inhibit the growth and induce cell death of FLT3-driven leukemia cells. AC220 is currently being evaluated in a phase II clinical trials in relapsed or refractory AML patients. 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 3619.

Activating mutations in the receptor tyrosine kinase FLT3 are present in approximately 30% of patients (pts) with acute myeloid leukemia (AML), and they have a significantly worse prognosis than pts with wild type (WT) FLT3, suggesting that the activated kinase is a driver of the disease and a potential target for kinase inhibitor therapy. AC220 is a novel 2nd generation class III receptor tyrosine kinase (RTK) inhibitor with potent in vitro and in vivo activity in FLT3-dependent tumors. It is highly selective for WT and mutant FLT3 and several other class III RTKs, including KIT, CSF1R, RET and PDGFR. AC220 is in a first-in-human phase 1 study for relapsed or refractory AML pts, unselected for FLT3 mutations. The study has a standard 3+3 dose escalation design with 50% dose increments. AC220 is administered once daily as an oral solution for 14 days followed by a 14 day rest period (1 cycle) with a starting dose of 12 mg. Concurrently, pts are being dosed on a continuous dosing regimen starting at 200 mg/day for 28 days (1 cycle). Pts with clinical benefit may continue to receive further cycles. Currently, 52 pts have been dosed with AC220 up to 450 mg/day (10 dose cohorts). Median age was 60 yrs (range, 23 to 86 yrs), median number of prior therapies was 3 (range, 0 to 8) and 2 pts had prior allogeneic hematopoietic stem cell transplant (HSCT). Two elderly patients (age ≥ 78 yrs) unfit for induction chemotherapy were previously untreated. Fifteen patients have FLT3 mutations (12 ITD and 3 TKD), 25 are WT, and 12 are undetermined. Pts are also evaluated for PK, pFLT3, pSTAT5, FLT3 genotyping and ex vivo plasma inhibitory activity. AC220 is well tolerated and MTD has not yet been observed with either schedule. One pt had a possibly drug-related DLT in the 18 mg cohort (grade 3 CHF, although pt had a pre-existing heart condition) leading to cohort expansion, but no other cases of drug-related CHF or other DLT have been seen. Other possibly drug-related AEs (most frequently gastrointestinal events) were mild (grade ≤ 2). Response data based on investigator’s assessment are available on the first 45 pts. Responses were observed in 11 (24%) pts. Four pts achieved a complete response (CR) – 2 with incomplete platelet recovery (CRp) and 2 with incomplete platelet and neutrophil recovery (CRi), one of these pts also had complete resolution of leukemia cutis. In addition, 7 pts had partial responses (PR, defined as a decrease of ≥ 50% blasts to levels of 5%–25% in the bone marrow). Most responses (8/11, 73%) occurred after cycle 1 and one was observed after cycle 3. Median duration of response is 18 weeks (range, 4 to 26+ weeks). Three responders are FLT3 mutants (2 ITD and 1TKD), 5 are WT, 3 are undetermined. Six of the 9 non-responding pts with ITD mutations had initial rapid clearing of peripheral blasts with intermittent AC220 dosing, but subsequently progressed or had disease-related mortality. All these pts had aggressive disease and received a median of 6 prior treatment regimens (range, 3 to 8). AC220 plasma exposure is sustained between dose intervals and continues to increase in a dose-proportional manner from 12 mg to 300 mg. FLT3 phosphorylation is strongly suppressed when plasma obtained from study pts is tested ex vivo in FLT3-ITD and WT cell lines at 12 mg and 60 mg doses, respectively. Assessments of pFLT3 and pSTAT5 from treated pts’ peripheral blood are ongoing and will be presented. Encouraging preliminary efficacy results and an acceptable safety profile warrants continued evaluation of AC220 as a single agent and in combination with other therapeutics for the treatment of AML.

Several different FLT3 inhibitors have now been tested as monotherapy for AML patients and have met with only limited success. To date, none of these inhibitors have resulted in complete, sustained inhibition of phosphorylated FLT3 in vivo, as measured by immunoblotting techniques. AC220 is a novel FLT3 kinase inhibitor with greater potency and kinase selectivity than others previously characterized. To establish a dose of AC220 that will lead to complete, sustained inhibition of phosphorylated FLT3, a Phase I open-label, sequential dose escalation study investigating safety, tolerability, pharmacokinetics, and pharmacodynamics in patients with relapsed or refractory acute myeloid leukemia is being conducted. AC220 is administered once daily as an oral solution for 14 days followed by a 14-day rest period, (one cycle) with a starting dose of 12 mg. Patients showing clinical benefit may continue to receive further cycles of therapy. The study is a standard 3+3 dose escalation design with 50% dose increments. Currently 52 patients have been enrolled and AC220 has been well tolerated and escalated to 450 mg daily (10th dose cohort) on this intermittent dosing regimen and PK has been evaluated up to 300 mg. AC220 is orally bio-available and has a long effective half-life, estimated to be 2.5 days, exhibiting minimal peak and trough variation of plasma levels. AC220 plasma exposure in AML patients is sustained between dose intervals and continues to increase in a dose-proportional manner from 12 mg to 300 mg daily, with steady-state plasma concentrations achieving greater than 1,500 nM at 300 mg. In addition, a pharmacologically active metabolite, AC886, has been identified in patient urine and plasma samples, which has similar potency and kinase selectivity to AC220 as determined by in vitro kinase binding and cell assays. The IC50s for AC220 and AC886 in FLT3-ITD-expressing cell lines and in primary FLT3-ITD AML patient blast samples are in the low nanomolar range (1.7 and 0.3 nM, respectively) and are in good agreement with cytotoxicity (Annexin V and MTT) assays. Both drugs display a high degree of selectivity for FLT3 in an IL-3 rescue assay. AC220 and AC886 also potently inhibit wild type FLT3 (3.6 and 4.2 nM, respectively). In standard immunoblot curves using AC220 and AC886 spiked into plasma, FLT3-ITD phosphorylation was completely inhibited at 50 and 200 nM, respectively. As predicted from the standard curve data, plasma from AC220-treated AML patients completely suppresses FLT3 phosphorylation on Day 1 and at steady-state in an ex vivo plasma inhibitory activity (PIA) assay, even at the lowest dose of 12 mg daily. AC220, with its metabolite AC886, is a potent, selective inhibitor of both mutant and wild type FLT3. It is orally bio-available with sustained plasma exposure, and the pharmacological impact of AC220 is further enhanced by the presence of AC886. In vivo, therefore, AC220 is expected to completely inhibit FLT3-ITD at >100 nM (achieved at the lowest AC220 dose of 12 mg) and FLT3-WT at 500 nM (achieved by the 60 mg dose). AC220 is the most potent and selective FLT3 inhibitor tested using the PIA assay thus far, and, unlike other FLT3 inhibitors that have been assessed in this assay, is the first to completely suppress FLT3 phosphorylation ex vivo at doses that are easily achievable and sustainable in the clinic. Therefore, AC220 should be able to effect substantial and prolonged target inhibition in patients. This makes it an ideal compound with which to test the hypothesis that complete, sustained inhibition of FLT3 activity will lead to clinical benefit in FLT3 mutant AML patients.

Activating mutations in the receptor tyrosine kinase FLT3 are present in up to approximately 30 % of acute myeloid leukemia (AML) patients, implicating FLT3 as a potential target for kinase inhibitor therapy. While several compounds have been evaluated in the clinic as FLT3 inhibitors, including CEP-701 (lestaurtinib), PKC-412 (midostaurin), MLN-518 (tandutinib) and most recently sorafenib, none of these was originally developed as a FLT3 inhibitor. We report here the characterization of AC220, a novel compound that has been expressly optimized as a selective FLT3 inhibitor. We demonstrate that this second generation FLT3 inhibitor has a unique combination of high potency, exceptional selectivity, bioavailability, and pharmacokinetic properties compatible with once a day oral dosing. In an in vitro binding assay AC220 interacted with FLT3 with high affinity (Kd = 1.6 nM). In the FLT3 dependent human leukemia cell line MV4-11, which harbors a homozygous activating FLT3 internal tandem duplication (ITD) mutation, AC220 inhibited FLT3 autophosphorylation and cellular proliferation with subnanomolar potency. Inhibition of proliferation of a FLT3 independent cell line was several hundred-fold less potent, demonstrating cellular selectivity for FLT3. A biochemical screen against a panel of 402 kinase assays representing almost 80 % of human protein kinases revealed a highly focused and selective interaction pattern. The only targets with affinity for AC220 within 10-fold that for FLT3 were closely related class III receptor tyrosine kinases (KIT, PDGFR, RET, CSF1R), and the only targets with affinity within 100-fold that for FLT3 were four additional receptor tyrosine kinases (FLT1, FLT4, DDR1, VEGFR2). When orally administered to mice at a dose of 10 mg/kg, AC220 achieved a peak plasma concentration (Cmax) of 3.8 μM (2,100 ng/mL) within two hours of dosing. When corrected for plasma protein binding, the concentration of AC220 in plasma remained above the cellular IC50 for FLT3 inhibition 24 hours after dosing. Total exposure (AUC0-24 h) as well as Cmax increased proportionally with the administered dose from 0.1 to approximately 30 mg/kg. At higher doses, both Cmax and AUC0-24 h continued to increase, approaching a plateau above 100 mg/kg. In a FLT3-ITD-dependent MV4-11 tumor xenograft model, AC220 showed substantial, dose dependent efficacy when dosed at 1, 3 and 10 mg/kg orally once a day for 28 days. Tumors regressed at 3 and 10 mg/kg, and remained static at 1 mg/kg. In a follow-on study at the 10 mg/kg oral dose, tumor size was monitored for an additional 60 days after dosing was discontinued. By the end of the study eight complete responses and two partial responses were observed in the ten animals treated with AC220. AC220 also had activity in a leukemia tumor model at doses as low as 1 mg/kg given orally once a day. A direct comparison of AC220 with the first generation FLT3 inhibitors CEP-701, MLN-518, PKC-412, sorafenib and sunitinib revealed that the combination of potency, selectivity and pharmacokinetic properties is unique to AC220. AC220 is a second generation FLT3 inhibitor that has been explicitly optimized for the combination of properties believed to be required for the successful treatment of FLT3-dependent AML, and specifically to test the hypothesis that selective FLT3 inhibition will result in clinical benefit. AC220 is currently being evaluated in a phase I clinical trial in relapsed or refractory AML patients.