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The use of multikinase inhibitors (MKI) in oncology, such as sorafenib, is associated with a cutaneous adverse event called hand–foot skin reaction (HFSR), in which sites of pressure or friction become inflamed and painful, thus significantly impacting quality of life. The pathogenesis of MKI-induced HFSR is unknown, and the only available treatment options involve dose reduction or discontinuation of therapy, which have negative effects on primary disease management. To investigate the underlying mechanisms by which sorafenib promotes keratinocyte cytotoxicity and subsequent HFSR induction, we performed a transporter-directed RNAi screen in human epidermal keratinocytes and identified SLC22A20 (OAT6) as an uptake carrier of sorafenib. Further investigations into the intracellular mechanism of sorafenib activity through in situ kinome profiling identified the mitogen-activated protein kinase MAP3K7 (TAK1) as a target of sorafenib that induces cell death. Finally, we demonstrate that sorafenib induced keratinocyte injury in vivo and that this effect could be reversed by cotreatment with the OAT6 inhibitor probenecid. Collectively, our findings reveal a novel pathway that regulates the entry of some MKIs into keratinocytes and explains the basis underlying sorafenib-induced skin toxicity, with important implications for the therapeutic management of HFSR. Cancer Res; 76(1); 117–26. ©2015 AACR.

Introduction: Mutations in fms-like tyrosine kinase 3 (FLT3) are common genomic abnormalities in adult acute myeloid leukemia (AML). When FLT3 is present as a dominant internal tandem duplication (ITD) AML clone, it is associated with poor treatment outcome and short overall survival (OS). A phase 3 study with the multi-kinase (including FLT3) inhibitor midostaurin improved OS of FLT3+ AML prompting widespread use, yet little is understood about resistance pathways and synergistic pharmacologic targets. We conducted a genome-wide CRISPR-KO (loss-of-function) screen in a FLT3+ AML cell line treated with midostaurin, validated results in vitro and in vivo, and identified distinct findings relative to limited CRISPR screening previously reported (Cancer Res 77:4402, 2017) with a very selective FLT3-ITD inhibitor (quizartinib). Methods: CRISPR pooled screens (GeCKOv2 library, >500 cells/sgRNA) of midostaurin versus vehicle were analyzed via MAGeCK. Gene essentiality was derived from guide abundance after 7-day selection of transduced cells compared to plasmid levels. In vitro validation included shRNA knockdown and/or inhibition followed by proliferation and clonogenic assays in cell lines and primary AML blasts. NSG mice (n=7/group) engrafted with MOLM-13-luciferase cells were treated with vehicle, single agents, or combination and evaluated for disease burden (IVIS) and survival (Kaplan-Meier). Results: We first confirmed our screening approach correlated well with previously reported essential genes (Cell Rep 17:1193, 2016; Cancer Cell 33:386, 2017;Genome Biol 16:281, 2015). We then identified >500 genes (FDR Conclusion: FLT3 inhibitors represent the first targeted therapeutic to impact AML survival and herein we use genome-wide CRIPSR screening to demonstrate that resistance mechanisms of broad-spectrum kinase inhibitors are distinct from those previously reported with specific inhibitors (quizartinib). Furthermore, we validate the select target BCL2 genetically and with the therapeutic venetoclax demonstrating in vitro and in vivo synergy, thereby justifying pursuit of this combination in future clinical trials. Ongoing efforts include validation of other targets that will be presented. Citation Format: Lindsey T. Brinton, Katie Williams, Shelley Orwick, Larry Beaver, Daniel Canfield, Casey Cempre, Jordan Skinner, Ronni Wasmuth, Quais Hassan, John C. Byrd, Rosa Lapalombella, James S. Blachly. Synthetic lethal and resistance pathways in midostaurin-treated AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 371.

Acute Myeloid Leukemia (AML) is a rapidly progressing hematopoietic malignancy arising from bone marrow myeloid progenitor cells. Treatment with cytotoxic chemotherapy has not changed for over four decades resulting in poor survival. The dismal prognosis could be attributed to the heterogeneity of this disease, where multiple genetically aberrant clones exist within the same patient. Mutations in the FMS-like tyrosine kinase (FLT3) occurs in 30% of AML patients, typically as an internal tandem duplication (ITD) resulting in a constitutively active FLT3 survival pathway. This has prompted the generation of selective FLT3 inhibitors such as Quizartinib and Gilteritinib which are currently being pursued in clinical trials. Still, acquired resistance to these selective FLT3 inhibitors due to the acquisition of tyrosine kinase domain mutations (TKD) can occur. This suggests that the use of an agent with a broader kinome inhibition profile (such as the recently granted FDA approved Midostaurin) could achieve more durable clinical benefit. ARQ 531 is a novel potent BTK inhibitor currently being investigated in a Phase 1 trial in patients with relapsed/refractory hematological malignancies (ClinicalTrials.gov Identifier: NCT03162536). We have found that ARQ 531 also has inhibitory activity against members of the Src family of kinases (SFK; including downstream target SYK) as well as FLT3. SYK directly binds to and trans-activates FLT3 which is essential for FLT3-ITD tumorigenicity, suggesting that ARQ 531 has therapeutic potential in AML. Therefore we have investigated the in vitro and in vivo efficacy of ARQ 531 in AML. Our preliminary studies demonstrate cytotoxicity for ARQ 531 in patient-derived primary AML cells harboring FLT3 wild type and FLT3-ITD, as well as multiple AML cell lines. Importantly, ARQ 531 is effective in a MOLM-13 tyrosine kinase inhibitor (TKI) resistant cell line harboring a FLT3-ITD-TKD-D835Y mutation. Furthermore, we show that ARQ 531 can reduce the level of phosphorylated FLT3, but unlike selective FLT3 inhibitors, it can also inhibit Src family phosphorylation and SYK phosphorylation. Additionally, ARQ 531 exhibited an anti-clonogenic effect on primary patient blasts using Methocult colony forming unit assay. Finally, to investigate the in vivo effect of ARQ 531, we used an aggressive AML MOLM-13 disseminated xenograft mouse model. NSG mice were randomized one-week post engraftment to either vehicle or daily oral gavage of 50 mg/kg ARQ 531. The estimated median survival for the ARQ 531 group was 23 days compared to 21 days for the vehicle group (p = 0.002) suggesting in vivo efficacy for ARQ 531 in AML. Collectively, we provide for the first time promising preclinical efficacy for ARQ 531 in AML supporting further mechanistic investigation of this agent, and potentially, expansion of the ongoing clinical studies to include AML patients. E. H. and J.C. B. contributed equally as co-senior authors to this work Citation Format: Ola A. Elgamal, Bridget Carmichael, Amy Lehman, Shelley J. Orwick, Minh Tran, Virginia M. Goettl, Shaneice Mitchell, Rosa Lapalombella, Jae Yoon Jeon, Sharyn D. Baker, Sudharshan Eathiraj, Brian Schwartz, Erin Hertlein, John C. Byrd. Preclinical evaluation of the tyrosine kinase inhibitor ARQ 531 in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1882.

Acute myeloid leukemia (AML) is the most prevalent adult leukemia characterized by genetic or mutational disruption of myeloid differentiation, growth arrest, and apoptosis. Standard “7 + 3” chemotherapy consisting of cytarabine in combination with an anthracycline such as daunorubicin has been used for more than four decades. However this course only results in 10 year disease free survival in 15% of patients age 60 years. Several new successful therapies in AML are shown to work by promoting differentiation, and/or inducing apoptosis of the terminally differentiated cells. However these therapies, while promising, have the limitation of working in only a subset of AML with specific genetic profiles. The discovery of novel agents applicable to a broader patient population still represents a critical unmet need in this disease. Intracellular calcium is a common signaling molecule used in a variety of cellular processes including cell cycle progression. One of the major pathways which regulates calcium entry is store operated calcium entry (SOCE) mediated through calcium release-activated Ca2+ channels (CRAC), which respond to depleted calcium stores in ER lumen. This primarily occurs via interaction of ORAI1 (expressed on the plasma membrane) and STIM1 (expressed on the endoplasmic reticulum). Leukemic cells in particular often rely on the influx of calcium through CRAC for proliferation, therefore targeting these channels are a promising therapeutic option. In the current study, we explore the preclinical use of RP4010, a novel inhibitor of ORAI1 developed by Rhizen Pharmaceuticals, in AML. RP4010 is very effective at inhibiting proliferation of both AML cell lines and primary AML patient samples (IC50 values ranging from 0.2 - 1 μM using a tetrazolium-based colorimetric assay (MTS) assay). Importantly, RP4010 efficacy is not altered in the presence of stromal cell support. RP4010 also decreased colony growth in CFU assays. Finally, we evaluated the in vivo efficacy using an MV4-11 xenograft model. RP4010 treatment at 50 mg/kg once daily by oral gavage significantly prolonged survival compared to the vehicle control (p = 0.019). Furthermore, at the time of sacrifice, splenic tumor burden (percentage of human CD45 positive cells) was reduced in the mice treated with RP4010 compared to the vehicle group (p = 0.019). In conclusion, we provide here compelling evidence that inhibition of ORAI1 with RP4010 is a promising therapeutic strategy in AML. A Phase 1/1b study in R/R Non-Hodgkin Lymphoma (NHL) patients is currently underway in the US (ClinicalTrials.gov Identifier: NCT03119467), and our data would support expansion to other hematological malignancies as well, particularly AML. Senior authors J. C. B and E. H contributed equally to this work. Citation Format: Bridget Carmichael, Amy Lehman, Ola A. Elgamal, Shelley J. Orwick, Jean Truxall, Larry Beaver, Kumar V. Penmetsa, Srikant Viswanadha, John C. Byrd, Erin Hertlein. Store-operated calcium signaling is an effective therapeutic target in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1938.

Enhancer of Zeste Homolog 2 (EZH2) is a catalytic core subunit of the Polycomb repressive complex 2, PRC2. EZH2 catalyzes the tri-methylation of lysine 27 on histone H3 (H3K27me3) which often results in the silencing of associated gene promoters. The over-expression of EZH2 is associated with cancer progression and poor prognosis in solid tumors and hematological malignances. EZH2 mutations in lymphoma generally are activating whereas those in myelodysplasia and acute myeloid leukemia are typically loss of function. Additionally, EZH2 mutations are not isolated to a hotspot and therefore discerning phenotype is challenging. The purpose of this study was to determine if the inhibition of EZH2 induces cell differentiation or apoptosis in AML, and to determine the significance of EZH2 mutations in AML patients. As H3K27me3 deposition is catalyzed by EZH2, we hypothesized that analysis of H3K27me3 levels could differentiate an inactivating EZH2 mutation. We analyzed H3K27me3 levels in AML patient samples with wild type (N = 5) and mutant (N = 3) EZH2, and found that overall H3K27me3 is reduced in the mutant patient samples. Furthermore, the sample with the highest variant allele frequency (VAF) of the EZH2 mutation had noticeably lower levels of H3K27me3 compared to those that had a lower VAF. This finding demonstrates that diminished H3K27me3 levels can predict the presence of an inactivating EZH2 mutation in AML cells. We next assessed if the EZH2 tool molecule EPZ011989 produced a similar phenotype in wild-type EZH2 AML cell lines (Kasumi-1, MOLM-13, and MV4-11). EPZ011989 inhibited H3K27me3 in all of the cell lines at a concentration of 0.625 μM after only four days of treatment. At these concentrations and this time point we demonstrated no change in viability among these three cell lines and only minimal proliferation inhibition in MV4-11 and MOLM-13 cells. Longer time points are currently being investigated as EZH2 inhibitors often induce anti-proliferative effects only after longer incubation periods. Therefore, our results support that inactivation of EZH2 with EZP011989 in AML cell lines induces the same cell-biochemical consequence (i.e. H3K27me3 loss) associated with EZH2 mutations in AML patients. This potentially will allow diverse pharmacologic studies in cell lines relative to gene targets and also synergistic lethality studies utilizing genetic and pharmacologic screening techniques to potentially develop therapeutic agents to treat this subtype of AML. Citation Format: Sydney Fobare, Cecelia Miller, Shelley J. Orwick, Heike Keilhack, Erin Hertlein, John C. Byrd. The effect of EPZ011989, an enhancer of Zeste homolog 2 inhibitor, in acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4520.

While dramatic improvements in survival have been achieved for children and adolescents with acute myeloid leukemia (AML), some subtypes are associated with event free survival of 8000 compounds. Compounds with >50% activity (N = 285) in the primary screen and known clinical candidates were prioritized for a secondary HTS performed in a dose-response manner (10 point curve, 0-10μM). Screening was performed using a 348-well plate format and a Biomek automation workstation for drug delivery. At 72 h cell viability was determined using Cell Titer Glo and automated Envision plate reader. Multiple drug classes had broad activity including: anti-malarials, cytotoxics (antimicrotubules, purine/pyrimidine antimetabolites, floxuridine, trimetrexate, topoI/II inhibitors), epigenetic modifiers (BET, DNMT and HDAC inhibitors), and agents targeting Bcl-2, Chk1, Parp, Hsp90, NF-êB, NAMPT, p53, proteasome, and Wee1. We validated 13 compounds from select drug classes (artesunate, cytarabine, gemcitabine, cabazitaxel, depsipeptide, panobinostat, vorinostat, ABT-199, RG117, bortezomib, carfilizomab, BMN673, MK-1775) using primary blast samples representing 3 high-risk groups (FLT3-ITD-positive, MLL-rearranged, FAB M7). Given the broad activity of gemcitabine and cabazitaxel across AML subtypes and the ability for repurposing in childhood AML, we further evaluated these agents alone and in combination in vitro. After 72h exposure, cabazitaxel was a potent inhibitor of viability in AML cell lines (IC50, 3-11nM) and primary blast samples (IC50, 3.4nM to >10μM); gemcitabine had similar activity (IC50, 3-62nM and 102nM to >10μM, respectively). The combination was evaluated using continuous and sequential administration schedules for up to 72h of treatment in several cell lines; simultaneous treatments were synergistic (CI, 0.2-0.45) and sequential treatments were additive to synergistic (CI, 0.82-0.95). Tolerable combination regimens were identified in NSG mice (5mg/kg cabazitaxel + 50mg/kg gemcitabine q3d x 4 weeks or q4d x 3 weeks) and efficacy studies in AML xenografts and primagrafts are ongoing. Further studies will aim to determine the underlying mechanisms of AML sensitivity to gemcitabine, cabazitaxel, and the combination. Our comprehensive approach to identify treatment leads provide advances in understanding the biology of a heterogeneous disease and development of novel treatment strategies with potential clinical benefit for pediatric AML. Citation Format: Christina D. Drenberg, Anang Shelat, Shelley J. Orwick, Hiroto Inaba, Raul C. Ribeiro, Jeffrey E. Rubnitz, Tanja A. Gruber, R K. Guy, Sharyn D. Baker. Integrated high-throughput screen to identify treatment leads for pediatric AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4786.

Improvements in survival have been achieved for children and adolescents with acute myeloid leukemia (AML), with the 5-year survival rate increasing between 1975 and 2010 from T), a gene encoding the hepatocellular uptake transporter OATP1B1. This SNP was not correlated with gene expression in leukemic blasts (P = 0.58), suggesting the association might be due to a host rather than a leukemia effect. Since this SNP is in strong linkage disequilibrium (P Citation Format: Christina D. Drenberg, Stanley Pounds, Lei Shi, Shelley Orwick, Lie Li, Shuiying Hu, Alice Gibson, Raul Ribeiro, Jeffrey Rubnitz, Alex Sparreboom, Sharyn D. Baker. Host variation in OATP1B1 is associated with treatment outcome in pediatric AML. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5464. doi:10.1158/1538-7445.AM2015-5464

Dasatinib, an orally available multikinase inhibitor, has been recently approved for imatinib- resistant chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. As with other tyrosine kinase inhibitors, dasatinib exhibits extensive interindividual pharmacokinetic variability, the causes of which are presently unknown. Previously, a notable degree of variability was observed with dasatinib oral absorption (time to peak concentration ranging from 0.5 to 6 h) and overall systemic exposure (CV, ∼50%) in patients (Demetri et al, Clin Cancer Res 2009). Identifying the factors underlying this variability may help enable clinicians to better manage the balance between dasatinib efficacy and toxicity. Here, we evaluated the role of ABCC4 (MRP4), a member of the ATP-binding cassette transporter family, as a factor regulating the oral absorption of dasatinib. Results from ABCC4-overexpressing inside-out vesicles indicated that dasatinib, at clinically relevant concentrations, was actively transported by ABCC4. Saturation of ABCC4-related transport was not seen even at a dasatinib concentration of 65 µM, suggesting high capacity transport. Dasatinib (1 µM) accumulation was increased ∼3-fold after a 5-min incubation in ABCC4-overexpressing vesicles compared to empty vector controls. The encouraging in vitro results prompted us to assess the in vivo role of ABCC4 in dasatinib absorption using Abcc4 knockout mice on a C57BL/6 background (Abcc4(-/-) mice) (Leggas et al, Mol Cell Biol 2004). Oral administration of dasatinib (10 mg/kg, in 50 mM sodium acetate buffer pH 4.6) to both wildtype mice and Abcc4(-/-) mice resulted in significant differences (P = 0.015) in dasatinib plasma concentrations at 4 out of 6 samples collected at serial time points. The largest difference was observed at 15 min, where dasatinib plasma concentrations in the Abcc4(-/-) mice were 5-fold decreased compared with wildtype mice. Although later time points also showed a decrease in dasatinib concentrations in Abcc4(-/-) mice (3.2-fold at 30 min; 2.2-fold at 1 h; 1.5-fold at 2 h), there was a convergence of the plasma curves by 4 h, suggesting that absorption rather than elimination pathways of dasatinib were altered in the Abcc4(-/-) mice. Indeed, pharmacokinetic analyses revealed a nearly 12-fold (P = 0.014) decrease in the dasatinib absorption rate constant in Abcc4(-/-) mice compared with wildtype mice, whereas the terminal half-life was unchanged (P = 0.41). The reduced absorption of dasatinib in Abcc4(-/-) mice was associated with a 4-fold (P = 0.019) decrease in peak concentration and a 2-fold decrease in AUC (P = 0.041). Collectively, these findings demonstrate that ABCC4 plays an important role in the intestinal absorption of dasatinib and reveal a new host factor that may contribute to enhanced interindividual pharmacokinetic variability in 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 1528.