FDA-approved Tyrosine Kinase Inhibitors Research Articles

Abstract A157: Antitumor activity of tarloxotinib, a hypoxia-activated EGFR TKI, in patient-derived lung cancer cell lines harboring EGFR exon 20 insertions

Abstract Tarloxotinib (TRLX) is a prodrug that releases an irreversible EGFR/HER2 tyrosine kinase inhibitor (TRLX-TKI) under pathophysiologically hypoxic conditions. Non-small cell lung cancer (NSCLC) has been characterized as a hypoxic disease and approximately 15% of lung adenocarcinomas harbor EGFR mutations. While most EGFR mutations predict for response to several FDA-approved tyrosine kinase inhibitors, in-frame insertions in exon 20 of EGFR are activating mutations in the tyrosine kinase domain that have significantly decreased sensitivity to EGFR inhibitors and currently have no approved targeted therapies. We derived and characterized three human lung adenocarcinoma cell lines with different EGFR exon 20 insertions in order to accelerate development of targeted therapies for this mutation class. Using these novel cell lines, we evaluated tarloxotinib as a therapeutic agent for tumors harboring this type of mutations. We demonstrate that our three patient-derived cell lines: CUTO14 (p.A767_V769dupASV), CUTO17 (p.N771_H773dupNPH), and CUTO18 (p.S768_770dupSVD) are dependent on EGFR for cell proliferation using shRNA-mediated knockdown. Our results show that EGFR exon 20 insertion cell lines are resistant to gefitinib; however, treatment with afatinib or TRLX-TKI reduces cell proliferation and signaling in a similar manner. The IC50 values for the three cell lines were 203nM, 89nM, and 709 nM for afatinib and 208nM, 33nM, and 345nM for TRLX-TKI, respectively. The prodrug form of tarloxitinib has minimal effect on cell proliferation in these models, consistent with the necessity for hypoxia-induced activation (to TRLX-TKI). Importantly, we evaluated the effect of tarloxotinib in vivo using murine xenograft models of CUTO14 and CUTO17. After four weeks of treatment, afatinib did not alter tumor growth compared to untreated tumors, whereas treatment with tarloxotinib induced significant tumor regression. The in vivo data suggest that the activated TKI of tarloxitinib is accumulating to biologically active concentrations in tumors following cleavage of tarlxoxotinib under hypoxic conditions. We conclude that our EGFR exon 20 insertions cell lines represent novel models for the investigation of therapeutic strategies for this mutation class. These cell lines have the ability to develop tumors in vivo and show reduced sensitivity to current EGFR TKIs, mimicking the lack of response in patients with these mutations. Finally, we demonstrate that tarloxotinib can overcome intrinsic EGFR exon 20 mutation resistance to standard EGFR TKIs. Citation Format: Adriana Estrada-Bernal, Andrea E. Doak, Anh T. Le, Hengbo Zhu, Nan Chen, Shevan Silva, Jeff B. Smaill, Adam V. Patterson, Robert C. Doebele. Antitumor activity of tarloxotinib, a hypoxia-activated EGFR TKI, in patient-derived lung cancer cell lines harboring EGFR exon 20 insertions [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 A157.

Abstract A141: Preclinical evaluation of a new ROS1 inhibitor to overcome resistance to EGFR therapy in glioblastoma

Abstract Glioblastoma (GBM) is the most aggressive brain tumor in adults and remains incurable despite multimodal intensive treatment regimens. The majority of GBM tumors show a mutated or overexpressed epidermal growth factor receptor (EGFR); however, tumors treated with EGFR inhibitors such as gefitinib or erlotinib inevitably recur. This recurrence highlights the need to identify signaling pathways involved in GBM resistance to EGFR therapy that may serve as targets for intervention. In our recently published work, we found that activation of the ROS1 pathway is a primary mechanism by which glioma cells become resistant to EGFR-targeted therapy. ROS1 is a proto-oncogene receptor tyrosine kinase activated by chromosomal rearrangement in several human cancers, including non-small cell lung cancer (NSCLC), cholangiocarcinoma, gastric cancer, ovarian cancer, and glioblastoma. We therefore designed, synthesized, and screened a series of new ROS1 inhibitors. The lead compound, ROS-Ic, is very potent (IC50 = 24nM) and highly specific to ROS1 protein. Treatment of gefitinib-resistant GBM cells with ROS1-lc sensitized the resistant cells to low concentrations of gefitinib, resulting in apoptosis following a prolonged S-phase cell cycle arrest. When compared to crizotinib, an FDA-approved small-molecule tyrosine kinase inhibitor currently used to treat ALK- and ROS1+ NSCLC patients, ROS-Ic showed superior cytotoxic activity in vitro towards ROS1-positive GBM cells. Crizotinib resistance is thought to be due to abolished crizotinib binding by ROS1 mutations in crizotinib binding pocket. Interestingly, the binding of our leading compound ROS1-Ic was not affected by these mutations and crizotinib-resistant cells harboring these mutations were still sensitive to ROS1-Ic, highlighting the potential therapeutic value of this compound. Citation Format: Hashim M. Aljohani, Mohamed Aittaleb, Byung Sun Park, So Ha Lee, El Mustapha Bahassi. Preclinical evaluation of a new ROS1 inhibitor to overcome resistance to EGFR therapy in glioblastoma [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 A141.

471TiP - A multicentre, randomised, double-blind, parallel-group, placebo-controlled trial of apatinib in local progressive or metastatic radioactive iodine-refractory differentiated thyroid cancer

Background: Radioactive iodine-refractory differentiated thyroid cancer (RAIR-DTC) is a big challenge in the management of thyroid cancer. Sorafenib and lenvatinib are the 2 FDA-approved tyrosine kinase inhibitors (TKIs), which might not be affordable for most of the Chinese patients (pts). Apatinib is an oral TKI targeting VEGFR-2, with a patient assistance program available in China. It achieved a quick Tg decline of 21% 2 weeks later and an objective response rate (ORR) of 90%, showing promising efficacy in RAIR-DTC (Lin et al, ATA 2016, Short Call Poster 65; Lin et al, Oncotarget, Epub Feb. 02, 2017). Thus, this study aimed to further evaluate the efficacy and safety of apatinib in treating RAIR-DTC. Trial design: This study is a multicentre, randomised, double-blind, parallel-group, placebo-controlled, phase III trial in China. Adult pts with locally advanced or metastatic RAIR-DTC are eligible. The inclusion criteria include at least one measurable lesion; disease progression within the past 12 months; and ECOG PS 0–2. Pts are defined as RAIR-DTC if they have target lesion(s) without iodine uptake, received one RAI treatment (≥3.7 GBq [≥100 mCi]) but progressed within the past 12 months, received two RAI treatments or more with a time interval of less than 12 months and progressed at least 12 months later), or received cumulative RAI activity over 22.2 GBq (≥600 mCi). Previous targeted therapy is not allowed. Enrolled patients will be randomly assigned to receive apatinib (500 mg qd) and placebo, respectively. Four weeks is defined as one cycle. Dose increase to 750 mg and dose reduction to 250 mg are allowed. The primary endpoint is progression free survival. The secondary endpoints include disease control rate, ORR, duration of response, changes in serum Tg and TgAb concentration, quality of life, and safety. A multiple Cox proportional hazards model is used to evaluate the hazard ratios after adjusting iodine uptake, metastatic lesion site, gender, and age. 118 pts will be recruited assuming a 106.9% increase in median PFS in the apatinib arm compared with the placebo arm. As of 2nd May 2017, 3 eligible patients have been enrolled. Clinical trial identification: NCT03048877 (Release date: February 7, 2017) Legal entity responsible for the study: Yansong Lin Funding: None Disclosure: All authors have declared no conflicts of interest.

Quantitative phosphoproteomic analysis of acquired cancer drug resistance to pazopanib and dasatinib

Acquired drug resistance impacts the majority of patients being treated with tyrosine kinase inhibitors (TKIs) and remains a key challenge in modern anti-cancer therapy. The lack of clinically effective therapies to overcome resistance represents an unmet need. Understanding the signalling that drives drug resistance will facilitate the development of new salvage therapies to treat patients with secondary TKI resistance. In this study, we utilise mass spectrometry to characterise the global phosphoproteomic alterations that accompany the acquisition of resistance to two FDA-approved TKIs, pazopanib and dasatinib, in the A204 rhabdoid tumour cell line. Our analysis finds that only 6% and 9.7% of the quantified phosphoproteome is altered upon the acquisition of pazopanib and dasatinib resistance, respectively. Pazopanib resistant cells display elevated phosphorylation in cytoskeletal regulatory pathways while dasatinib resistant cells show an upregulation of the insulin receptor/IGF-1R signalling pathway. Drug response profiling rediscovers several previously reported vulnerabilities associated with pazopanib and dasatinib resistance and identifies a new dependency to the second generation HSP90 inhibitor NVP-AUY-922. This study provides a useful resource detailing the candidate signalling determinants of acquired TKI resistance; and reveals a therapeutic approach of inhibiting HSP90 function as a means of salvage therapy to overcome pazopanib and dasatinib resistance. SignificancePazopanib and dasatinib are tyrosine kinase inhibitors (TKIs) approved for the treatment of multiple cancer types. Patients who are treated with these drugs are prone to the development of drug resistance and consequently tumour relapse. Here we use quantitative phosphoproteomics to characterise the signalling pathways which are enriched in cells that have acquired resistance to these two drugs. Furthermore, targeted drug screens were used to identify salvage therapies capable of overcoming pazopanib and dasatinib resistance. This data advances our understanding of the mechanisms of TKI resistance and highlights candidate targets for cancer therapy.

Imatinib Mesylate as an Effective Anti-viral Treatment for Alphavirus Infections

Alphaviruses are plus-strand RNA viruses that are transmitted by mosquitoes. There are very limited vaccines and treatment options available to those infected with alphaviruses, resulting in significant human and animal morbidity and mortality each year. Viruses are parasites of host cell metabolism and alphaviruses have been shown to increase glycolytic flux during infection to aid viral replication. Imatinib mesylate is an FDA-approved tyrosine kinase inhibitor that is used to treat several types of cancers. A hallmark of tumorous cells is an elevated metabolic rate and Imatinib successfully slows metabolism by inhibiting tyrosine kinases that are required to activate metabolic enzymes, such as hexokinase in the glycolytic pathway. It was hypothesized that Imatinib could be used to slow metabolism in virally-infected cells and reduce viral replication. Alphavirus-infected cells were treated with various concentrations of Imatinib and at a concentration of 6 µM, viral replication was reduced by more than 40% while cell viability was still at 100%. The efficacy of Imatinib treatment at inhibiting alphavirus replication was confirmed at different times post infection (6, 12, 18, and 24 hours post infection), different levels of infection (multiplicities of infection= 0.1, 1, and 10), and within different cell lines (BHK, Huh7 and HEK). Further analysis in mouse or other animal models is needed to confirm the utility of Imatinib as a therapeutic option for treating alphavirus infection, but the data are promising and shows a significant reduction in viral replication and may represent a novel treatment option for alphavirus infections.

Abstract 3146: Targeting ANXA3 in combination with sorafenib for the treatment of hepatocellular carcinoma

Abstract Sorafenib is the only FDA-approved tyrosine kinase inhibitor for targeted therapy in advanced HCC. Nevertheless, its efficacy is limited with only a modest improvement in patient outcome, likely due to acquired resistance. In-depth understanding of the molecular mechanism of sorafenib resistance is warranted for the development of novel treatment strategies. Recent studies by us and others have characterized liver tumor-initiating cells (T-ICs) to be a possible source of resistant and recurrent tumors and a plausible target for HCC treatment. Our group has previously identified CD133 to be a functional marker of liver T-ICs and found annexin a3 (ANXA3) to regulate cancer and stem cell-like properties in this subset of cells. Interestingly, our recent observations also found CD133+ liver T-ICs to be more resistant to sorafenib. Sorafenib resistant clones, established in HepG2 and Huh7 cells by continuous exposure to increasing concentrations of sorafenib, displayed enhanced abilities to migrate, invade, self-renew, and initiate tumor formation in immunodeficient mice, as well as higher expression of stemness associated genes. These two sorafenib resistant cell lines and two other sorafenib resistant HCC patient-derived xenografts established in a similar manner were also found to be enriched for CD133 and ANXA3 expression. Sorafenib resistant clones with ANXA3 stably suppressed were re-sensitized to sorafenib treatment and had diminished ability to migrate, invade, self-renew and initiate tumor growth in vivo, further substantiating the role of ANXA3 in mediating sorafenib resistance in HCC. Mechanistically, an activated PKC/ERK/FRA2 signaling axis was found to be responsible for driving this phenomenon. Clinically, ANXA3 expression was also found to have prognostic value as a higher ANXA3 expression in HCC patients who have received sorafenib treatment was correlated with poor overall survival. The combinatorial use of a homemade ANXA3 neutralizing antibody and sorafenib on HCC patient derived xenografts is now being investigated as a potential new treatment regimen for combating sorafenib resistance in HCC. Citation Format: Man Tong, Steve Luk, Noelia Che, Jin Ding, Terence KW Lee, Stephanie Ma. Targeting ANXA3 in combination with sorafenib for the treatment of hepatocellular carcinoma [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 3146. doi:10.1158/1538-7445.AM2017-3146

Identification of Targeted Therapies for Rare Adult Mature T-Cell Leukemia Using Functional Ex Vivo Screening of Primary Patient Samples

Introduction: T-cell prolymphocytic leukemia (T-PLL) is an aggressive lymphoproliferative disorder and accounts for ~2% of cases of mature lymphocytic leukemia in adults. The sparsity of patients with this disease poses a challenge to effectively develop targeted treatments. Recent studies suggest chromosome 14 abnormalities are most common in T-PLL (75% of patients), resulting in overexpression of the proto-oncogene TCL1 and activation of protein kinase B (Stengel, Anna, et al. 2016). Presently alemtuzumab is the only effective, available therapeutic option, with almost all patients relapsing within months after completion of therapy. With the aim of identifying new therapeutics for T-PLL, we assessed the sensitivity of primary patient samples to various targeted agents using an ex vivo functional screening platform.Methods: We have previously screened over 1000 primary specimens from patients with various hematologic malignancies against a panel of 143 single-agent small-molecular inhibitors, including kinase inhibitors, bromodomain inhibitors, BHM mimetics, proteasome inhibitors, IDH1/2 inhibitors, and several other drug classes. Primary patient specimens were cultured in the presence of each drug in a 7-point dose series. Sensitivity was assessed by a tetrazolium-based viability assay on day 3. An IC50 value was then calculated for each inhibitor and compared with the median of the IC50 values for the full patient sample cohort screened to date. Inhibitors for which the IC50 value was < 20% of the cohort median were considered effective. T-PLL primary patient samples were assessed for sensitivities using this historical drug sensitivity data and we ranked all drugs by their IC50.Results: Our database identified 5 adults (3 male, 2 female) with T-PLL who had analyzable data obtained within 1 month of diagnosis (Table 1). Median age was 65 years (range, 49-88 years). At diagnosis, the median white blood cell count was 56.2 K/cu mm (16-243.65 K/cu mm) and all patients had detectable 14q11 abnormalities and TCR gene rearrangements. All patients were treated with standard first-line therapy with IV alemtuzumab (30 mg 3x/week) for an average of 11.6 weeks (8.59 -14.75 weeks). Their median progression free survival (PFS) was 9.3 months (5.4-20 months) and duration of response was 5.6 months (2.9 -16.8 months). Three patients received second-line therapy after alemtuzumab failure with a PFS of 6.9 months (1.1- 9.8 months). Three patients went on to an allogeneic stem cell transplants with PFS of 6.7 months (4.5-7.7 months). The median overall survival was 19.1 months (2.07-11.6 months).Our ex vivo assay showed that 9 (6.2%) of 143 small-molecular inhibitors were considered effective (20% of median IC50) in the majority of subjects. This included 6 FDA approved tyrosine kinase inhibitors (axitinib, dasatinib, sunitinib, sorafenib, pazopanib, and ponatinib), midostaurin, an NF-kB activation inhibitor, and CSF1R inhibitors. Sensitivity of T-PLL patients to these drugs is shown in the log plot (Figure 1). Patient 4 had no strong targets identified. Dasatinib showed the most efficacy in 4 of the 5 patients, with an average IC50 of 0.003 ± 0.0007 µm, followed by CSFIRJ inhibitor with an average IC50 of 0.02 ± 0.015 µm.Conclusions: Our functional ex vivo screen of primary patient samples allowed us to evaluate the effectiveness of several FDA approved and investigational therapeutic agents targeting multiple oncogenic pathways in T-PLL, a rare disease with limited existing therapies. Our approach indicates that several existing inhibitors may be effective in this aggressive leukemia and can be tested in prospective clinical trials. [Display omitted] [Display omitted] DisclosuresDruker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy.

Abstract 5188: Combination of radiofrequency ablation and Sunitinib in the treatment of experimental hepatocellular cancer

Abstract Radiofrequency Ablation (RFA) is one of current clinical option in the treatment of hepatocellular cancer (HCC); however, RFA monotherapy is often not curative as a consequence of tumor micro-metastases and recurrence. Thus, induction of systemic response with additive treatment is required. In the present study, we are exploring the combination of RFA and sunitinib, an FDA-approved multiple tyrosine kinase inhibitor, in the treatment of HCC. Using our established strategy a clinically relevant murine model of HCC is prepared which mimics human disease and reflect its typical features. The follow-up magnetic resonance imaging (MRI) is applied to monitor tumor progression until the cumulative tumor volume reaches 2000 mm3. Size-matched tumor-bearing mice are assigned into 3 groups and receive the following treatments: no treatment, RFA alone with our improved machine and optimized parameters, combination of sunitinib and RFA. The therapeutic efficacy is evaluated by measuring tumor volume with MRI every twice weeks. We demonstrate that tumors grow substantially in the mice without treatment. RFA alone effectively destroys tumors, but unable to prevent tumor recurrence and micro-metastasis. In contrast, &amp;gt; 50% size reduction in tumor size is detected in the combined group compared to that in control mice, even no detectable tumors are observed in some treated mice. In conclusion, combination of sunitinib and RFA powerfully suppress tumor growth and could be translate into clinic practice. Citation Format: Xiaoqiang Qi, Guangfu Li, Dai Liu, Kevin F Staveley-O’Carroll, Eric Kimchi. Combination of radiofrequency ablation and Sunitinib in the treatment of experimental hepatocellular cancer. [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 5188.

Abstract 4741: Targeting tumor growth in a medullary thyroid carcinoma mouse model using a combinatorial treatment with a tyrosine kinase inhibitor and histone deacetylase inhibitor

Abstract Neuroendocrine tumors are rare forms of cancers arising from hormone-producing cells that are scattered throughout the body. Medullary thyroid carcinoma (MTC) originates from the thyroid gland parafollicular cells. Treatment options for progressive, metastatic MTC patients are limited to the recently FDA-approved tyrosine kinase inhibitors (TKIs), Vandetanib and Cabozantinib, which target multiple receptor tyrosine kinases, including VEGFR2. However, the efficacy of TKIs is limited and development of TKI resistance is common. Here we examined, the effect of a combinatorial drug therapy using the TKI, Nintedanib, and the histone deacetylase (HDAC) inhibitor, Romidepsin, on MTC growth in the NSE/p25-gfp bi-transgenic mouse line, an inducible MTC mouse model (see Pozo et al. 2013). The TKI- and HDAC inhibitors were administered intraperitoneally for 3 weeks and tumor growth progression was monitored using a T2 weighted magnetic resonance imaging on a 7 Tesla system. Tumor tissues were analyzed for oncogenic signaling pathways by immunoblotting and immunohistochemistry. We find that co-administration of Nintedanib and Romidepsin stops tumor growth and increases the number of necrotic foci within the tumor tissue. Interestingly, Nintedanib treatment alone reduced tumor vascularization as shown by decreased density of the vascular marker, CD31, but did not stop parafollicular cell proliferation. Taken together, these results suggest that a combinatorial drug therapy with a TKI- and an HDAC inhibitor may be a more efficient strategy to target MTC progression and overcome recurrence. Citation Format: Karine Pozo, Stefan Zahler, Chunfeng Tan, Keisuke Ishimatsu, Masaya Takahashi, James Bibb. Targeting tumor growth in a medullary thyroid carcinoma mouse model using a combinatorial treatment with a tyrosine kinase inhibitor and histone deacetylase inhibitor. [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 4741.

Dasatinib affects focal adhesion and myosin regulation to inhibit matrix contraction by Müller cells

Epiretinal membrane (ERM) contraction is associated with a variety of ocular diseases that cause macular dysfunction. Trans-differentiated Müller cells have been identified in ERMs, and have been implicated to be involved in the contractile process. In this study, we tested the effect of dasatinib, an FDA-approved tyrosine kinase inhibitor, on matrix contraction caused by Müller cells, and examined molecular mechanism of action. Type I collagen matrix contraction assays were used to examine the effect of drugs on matrix contraction by trans-differentiated Müller cells. Fluophore-conjugated phalloidin was used for the detection of actin cytoskeleton, and Western-blot analyses were carried out to examine protein expression and phosphorylation status. Dasatinib inhibited collagen matrix contraction by trans-differentiated Müller cells that was associated with decreased cell spreading and reduction of actomyosin stress fibers. Concomitantly, dasatinib-treated Müller cells had reduced phosphorylation of Src family kinase, paxillin, as well as myosin II light chain. Specific inhibitors of Rho/ROCK and myosin II confirmed the critical role played by this pathway in Müller cell contraction. Our data demonstrate that dasatinib significantly reduced matrix contraction by Müller cells via inhibition of focal adhesion, as well as actomyosin contraction.

Genotyping and immunohistochemistry of gastrointestinal stromal tumors: An update.

Gastrointestinal stromal tumors (GISTs) were originally thought to harbor either KIT or platelet-derived growth factor receptor A (PDGFRA) mutations only. However, more recent discoveries have highlighted additional, less common oncogenic driver mutations including NF1, BRAF, and succinate dehydrogenase (SDH) mutations. Genotyping GISTs has become more important since not all genotypes respond equally to FDA-approved tyrosine kinase inhibitors. GIST is a paradigm for personalized cancer therapy. Recent studies demonstrate how immunohistochemistry can be used both to diagnose GIST and to screen for specific mutations. DOG1 is particularly useful in the diagnosis of KIT-negative GIST, including tumors with PDGFRA mutations, which can also potentially be identified by immunohistochemistry for PDGFRA. SDHB immunohistochemistry is useful in characterizing GISTs with SDHA-D mutations, whereas SDHA immunohistochemistry is able to identify SDHA mutant GISTs.

Detection of Compound BCR-ABL Mutations in TKI Resistant CML Patients Including a Novel K245N Mutation Associated with Primary Nilotinib Resistance By Employing a Newly Developed Cost Effective BCR-ABL Sequencing Protocol

Abstract Introduction: BCR-ABL mutations are the major background players in manifestation of resistance to all FDA approved tyrosine kinase inhibitors (TKIs) including imatinib, dasatinib, nilotinib, Bosutinib and ponatinib 1. Detection of mutations is a vital part of European or North American clinical guidelines at the time of resistance and/or drug switching, because resistance-causing mutations appearing as a result of one drug are sensitive to other in many instances and mutational data can therefore help in prescription of better alternative TKI in case of resistance or unsatisfactory response 1,2. Although BCR-ABL sequencing protocols are reported, they either lack the experimental details or are not cost-effective to be used in third world countries 1’3. Therefore, objective of this study was to develop a cost-effective protocol for BCR-ABL mutation detection in TKI resistant CML. Material and Methods: Peripheral blood samples were collected from 10 imatinib resistant, 10 imatinib sensitive, 5 CML patients receiving nilotinib, positive for Philadelphia chromosome by conventional cytogenetics, and 10 healthy volunteers. Isolation of RNA was performed using TriZol® LS reagent and complementary DNA (cDNA) was prepared using random hexamer primers. The integrity of cDNA was checked by amplification of housekeeping gene GAPDH. A nested RT-PCR assay was optimized for ABL kinase domain amplification using standard PCR optimization techniques. PCR bands of 1306 or 1380 base pairs, corresponding to b2a2 and b3a2 BCR-ABL splice variants, were detected in 25 CML patients but no healthy controls. Consumables for CDNA and PCR were used from Fermentas (USA). PCR products were purified using Quick gel extraction kit (Invitrogen). DNA sequencing was performed using BigDye® Terminator v3.1Cycle Sequencing Kit (Applied Biosystems). Results: Compound mutations were detected in CML patient showing primary resistance to nilotinib, including a novel K245N mutation and G250W mutations (Figure 1) while 4 of nilotinib responders did not show any mutations. Similarly, mutations detected in four (4/10, 40%) imatinib resistant were (G250W), (T394A), (Y253H), (E355G, Y393H}. Of 10 imatinib sensitive patients, mutations were detected in 3, 2 in accelerated phase and 1 in blast crisis, while none in 7 c chronic phase CML (Figure 2). Discussion: We show association of BCR-ABL mutations with imatinib/nilotinib resistance and disease progression in CML patients, which is in accordance with previous studies 1’2’4,5. This also proves the usefulness and applicability of our BCR-ABL sequencing protocol for detection of clinically relevant mutations in CML patients receiving TKI treatment. A cost effective protocol it will facilitate the incorporation of mutation detection in clinical setting in low-resourced laboratories from third world countries and thus help better manage clinical interventions in drug-resistant CML 6’7.

Abstract 3674: Targeted oncology therapeutics show unique cardiotoxic profiles: Ponatinib as a case study

Abstract Although tyrosine kinase inhibitors (TKI) have greatly improved the treatment and prognosis of multiple cancer types, unexpected clinical cardiotoxicity has occurred with some of these agents that was not predicted by standard preclinical toxicity assessment methods. Since cardiotoxicity with TKI are often caused by the inhibition of signaling pathways that are critical to cardiac cell function, multi-targeted TKI that block multiple pathways increase the likelihood for cardiac cell damage. We recently demonstrated that an in vitro multi-parameter test panel assessing the effect of drug treatment on overall cardiac health and function could accurately predict the cardiotoxicity of four FDA-approved TKI. The multi-targeted TKI that are clinically associated with cardiac adverse events (crizotinib, sunitinib, nilotinib) all proved to be cardiotoxic in our in vitro tests while a more targeted and relatively cardiac-safe drug, erlotinib, showed only minor changes in cardiac cell health. The present studies expand upon this initial observation using a broad panel of drugs such as TKI (including the recently withdrawn TKI ponatinib), chemotherapeutic agents, and non-oncology compounds, which were grouped based on their known clinical toxicity profiles ranging from cardiac safe to withdrawn. We assessed each compound's cardiotoxic potential by examining their effect on cell viability, reactive oxygen species (ROS) generation, lipid formation, troponin secretion and beating activity in a human induced pluripotent-derived cardiac stem (iPS) cell model. The results showed a unique cardiotoxic signature of oncology therapeutics that was significantly different from other drug classes. Oncology compounds, including ponatinib, induced metabolic stress (ROS generation and lipid formation) resulting in potent cardiac cell death and troponin secretion while other classes of drugs disrupted cardiac cell beating with minimal impact on cardiac cell health. In comparison, cardiac-safe drugs, including non-oncology compounds and more targeted TKI, did not impact any of the endpoints that assessed cardiac cell health and function. Our results demonstrate unique toxicity profiles for different drug classes which may reflect multiple mechanisms of cardiotoxicity. For oncology compounds specifically, multi-targeted TKI and chemotherapeutic agents show potent cardiac toxicity while more targeted agents are less cardiac toxic. These studies show that a multi-parameter approach provides a comprehensive and robust assessment of cardiotoxic potential by evaluating several parameters that are important to cardiac cell health and function in a more predictive adult cardiac cell model. A panel such as this aids in early risk assessment of cardiotoxic compounds and may reduce drug failure due to toxicity discovered in later phases of development or after drug approval such as that shown with ponatinib. Citation Format: Dominique Talbert, Kimberly Doherty, Patricia Trusk, Diarmuid Moran, Scott Shell, Sarah Bacus. Targeted oncology therapeutics show unique cardiotoxic profiles: Ponatinib as a case study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3674. doi:10.1158/1538-7445.AM2014-3674

Abstract 2137: Prevention of HER2 overexpressing early stage breast disease progression by lapatinib

Abstract Currently, Tamoxifen and Aromatase inhibitors are used for prevention of estrogen receptor positive (ER+) breast cancer; however, there is no effective prevention strategy for ER- breast cancer. HER2 overexpression (HER2+) can be detected in up to 60% early stage breast lesions, such as atypical hyperplasia (ADH) and ductal carcinoma in situ (DCIS). Up to 40% of ER- breast cancers are HER2+. Thus, targeting HER2 signaling may be an effective chemoprevention strategy for these ER- and HER2+ breast cancer. Here, we stable introduced ErbB2-expressing retroviruses into the ER-, non-transformed MCF10A cells (MCF10A.B2) to establish an ER- and HER2+ early breast disease model. We show that Lapatinib, a FDA-approved dual tyrosine kinase inhibitor for both HER2 and EGFR, reduced the proliferation rate and increased the apoptosis in MCF10A.B2 cells. In 3D culture, Lapatinib treatment starting at day 6 prevented the MCF10A.B2 cells from forming abnormal acini, and the numbers of DCIS-like acini have been significantly decreased compared to control group. Untreated MCF10A.B2 cells forms DCIS-like acini in 3D culture by day 10, which have been reverted to normal or ADH-like acini when lapatinib treatment started by day 10. Furthermore, MMTV-neu mouse model develop ER- mammary tumors in 6.4 months, whereas lapatinib treatment (30mg/kg/day, six days per week) starting at 10 weeks of age significantly delayed the tumor initiation and progression in this mouse model as detected by mammary gland biopsy and fiber optic micro-endoscope (FOME) imaging. Mice treated with lapatinib had fewer premalignant lesions and reduced noninvasive tumors in their mammary glands, which are correlated with reduced proliferation rate and increased apoptosis rate, indicating that lapatinib is a promising agent for the prevention/intervention of ER- and HER2+ breast cancers. To identify effective and reliable biomarkers to assess the risk of ER-/HER2+ lesions and efficacy of lapatinib for prevention and intervention, cDNA microarray and reverse phase protein array (RPPA) were performed on MCF10A.B2 cells and control MCF10A cells. We have identified several molecular pathways activated by HER2+ in this model which will be tested for their roles in the initiation of mammary tumors and as potential targets for prevention of ER-/HER2+ breast disease. Our study brings better understanding of the mechanism underlying ER-/HER2+ early stage breast disease progression to cancer and could be rapidly translated into the clinic for prevention of ER-/HER2+ breast cancers. Citation Format: Jia Xu, Shalini Jain, Dihua Yu. Prevention of HER2 overexpressing early stage breast disease progression by lapatinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2137. doi:10.1158/1538-7445.AM2014-2137

Multi-parameter in vitro toxicity testing of crizotinib, sunitinib, erlotinib, and nilotinib in human cardiomyocytes

Tyrosine kinase inhibitors (TKi) have greatly improved the treatment and prognosis of multiple cancer types. However, unexpected cardiotoxicity has arisen in a subset of patients treated with these agents that was not wholly predicted by pre-clinical testing, which centers around animal toxicity studies and inhibition of the human Ether-à-go-go-Related Gene (hERG) channel. Therefore, we sought to determine whether a multi-parameter test panel assessing the effect of drug treatment on cellular, molecular, and electrophysiological endpoints could accurately predict cardiotoxicity. We examined how 4 FDA-approved TKi agents impacted cell viability, apoptosis, reactive oxygen species (ROS) generation, metabolic status, impedance, and ion channel function in human cardiomyocytes. The 3 drugs clinically associated with severe cardiac adverse events (crizotinib, sunitinib, nilotinib) all proved to be cardiotoxic in our in vitro tests while the relatively cardiac-safe drug erlotinib showed only minor changes in cardiac cell health. Crizotinib, an ALK/MET inhibitor, led to increased ROS production, caspase activation, cholesterol accumulation, disruption in cardiac cell beat rate, and blockage of ion channels. The multi-targeted TKi sunitinib showed decreased cardiomyocyte viability, AMPK inhibition, increased lipid accumulation, disrupted beat pattern, and hERG block. Nilotinib, a second generation Bcr-Abl inhibitor, led to increased ROS generation, caspase activation, hERG block, and an arrhythmic beat pattern. Thus, each drug showed a unique toxicity profile that may reflect the multiple mechanisms leading to cardiotoxicity. This study demonstrates that a multi-parameter approach can provide a robust characterization of drug-induced cardiomyocyte damage that can be leveraged to improve drug safety during early phase development.

Abstract 2040: Indication switching of the FDA-approved anticancer tyrosine kinase inhibitors to treat metabolic diseases.

Abstract Drug switching is a unique way to identify and extend new indications for approved drugs in drug discovery &amp; development. Tyrosine kinase inhibitors (TKIs) are used in biologically targeted cancer therapy and so far 12 TKIs have been approved by FDA. We hypothesize that these TKIs act on other molecular targets in addition to tyrosine kinases and may manage metabolic diseases given that there is a complex network of kinases that work together to regulate a number of important cellular processes. Employing a comprehensive docking method with our established chemical-protein interactome (CPI) and 11 FDA-approved TKIs, we have discovered301 PDB-deposited proteins corresponding to 353 ligand binding pockets among a total of 1,780 PDB-deposited human protein entries. Notably, sorafenib, dasatinib and crizotinib had a CPI binding score (ZZ_score) of -1.2903, -1.0278 and -1.5384 against histone deacetylase 7A, respectively. In addition, those TKIs achieved high ZZ scores against B-Raf, PPAR and VDR, suggesting a high binding affinity of sorafenib, dasatinib and crizotinib with these proteins. Our preliminary studies have showed that the acetylated-lysine in α-tublin and oncogenic Akt and Raf-signaling was inhibited significantly in human multiple melanoma cells by these TKIs. Interestingly, dasatinib increased VDRE and HDLR luciferase reporter activity in the human lung adenocarcinoma A-549 cells, and dasatinib and crizotinib induced autophage by activated LC3 in vitro. Furthermore, the erlotinib and sorafenib decreased glucose levels in Wistar STZ-D rats. These TKIs are predicted to act on a series of therapeutics targets associated with metabolic diseases (such as PPAR and Sirt1). Collectively, FDA-approved TKIs may be switched to become a “magic bullet” concurrently targeting tyrosine kinase, HDAC, PPAR, VDR and B-Raf, shedding a light for future anti-metabolic disorder and anticancer drug discovery &amp; development. Further validation of additional “hot targets” besides tyrosine kinase such as HDAC, B-Raf, PPAR, Sirt1, Akt and VDR, and in vivo evaluation of anti-metabolic disorder by TKIs are undergoing at our laboratory. These findings suggest that drug switching may represent a new and effective approach to expanding the application of existing drugs. Citation Format: Jiazhi Sun, Lun Yang, Minghua Li, Zhixin Wang, Amy Haynes, Qian Wang, Isaac Raplee, Jean Melchisedek, Anand Prasad, Kevin B. Sneed, Lin He, Shufeng Zhou. Indication switching of the FDA-approved anticancer tyrosine kinase inhibitors to treat metabolic diseases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2040. doi:10.1158/1538-7445.AM2013-2040

Abstract 4447: Nanoformulation of lapatinib for HER2-positive breast cancer patients with brain metastasis

Abstract Despite HER2-targeting agents having a reduced risk of distant relapse in early-stage breast cancer and improved systemic control in metastatic breast cancer, relapse or progression in the brain is a common and serious concern for mortality and morbidity in these HER2-positive breast cancer patients. Currently, radiation is the only effective therapy for these patients with various clinical outcomes. Thus, there is a significant unmet need to develop new therapeutic modalities to improve treatment efficacy for these patients, especially for patients who have failed radiation therapy. The objective of this project is to develop a novel nanoformulation of lapatinib for the treatment of HER2-positive breast cancer patients with brain metastasis. Lapatinib is the only FDA-approved small molecule tyrosine kinase inhibitor targeting both HER2 and epidermal growth factor receptor (EGFR). Lapatinib anecdotally induces objective response in HER2-positive breast cancer patients with brain metastasis. Current preclinical and clinic data suggest that lapatinib has limited and variable penetration through the blood-brain barrier, which is also affected by large inter-patient variations in the bioavailability of oral formulation of lapatinib. Dr. Lam's laboratory has developed a novel nanoparticle technology using oligocholic acid based micelles (telodendrimers) to load a high dose of hydrophobic drugs for cancer treatment. We have generated a stable nanoformulation of lapatinib with drug loading up to 10 mg per mL. The average particle size is less than 20 nm in diameter, which is desirable for tumor targeting due to the preferential drug accumulation at the tumor site via the enhanced permeation and retention (EPR) effect. We are currently optimizing and characterizing the physicochemical properties of several lapatinib-loaded nanoformulations. The most stable and efficacious nanoformulation of lapatinib will be selected for further preclinical testing using in vitro and in vivo models of HER2+ breast cancer. This nanoformulation of lapatinoib will be suitable for intrathecal injection as it is not affected by individual variations in oral bioavailability and penetration through the blood brain barrier. If successful, we would design a phase I/II clinical study to determine the safety and efficacy of this new nanoformulation of lapatinib for HER2+ breast cancer patients with refractory brain metastasis or meningeal disease. 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 4447. doi:10.1158/1538-7445.AM2011-4447

The lack of target specificity of small molecule anticancer kinase inhibitors is correlated with their ability to damage myocytes in vitro

Many new targeted small molecule anticancer kinase inhibitors are actively being developed. However, the clinical use of some kinase inhibitors has been shown to result in cardiotoxicity. In most cases the mechanisms by which they exert their cardiotoxicity are not well understood. We have used large scale profiling data on 8 FDA-approved tyrosine kinase inhibitors and 10 other kinase inhibitors to a panel of 317 kinases in order to correlate binding constants and kinase inhibitor binding selectivity scores with kinase inhibitor-induced damage to neonatal rat cardiac myocytes. The 18 kinase inhibitors that were the subject of this study were: canertinib, dasatinib, dovitinib, erlotinib, flavopiridol, gefitinib, imatinib, lapatinib, midostaurin, motesanib, pazopanib, sorafenib, staurosporine, sunitinib, tandutinib, tozasertib, vandetanib and vatalanib. The combined tyrosine kinase and serine-threonine kinase selectivity scores were highly correlated with the myocyte-damaging effects of the kinase inhibitors. This result suggests that myocyte damage was due to a lack of target selectivity to binding of both tyrosine kinases and serine-threonine kinases, and was not due to binding to either group specifically. Finally, the strength of kinase inhibitor binding for 290 kinases was examined for correlations with myocyte damage. Kinase inhibitor binding was significantly correlated with myocyte damage for 12 kinases. Thus, myocyte damage may be multifactorial in nature with the inhibition of a number of kinases involved in producing kinase inhibitor-induced myocyte damage.

Abstract 5604: Modulation of Stat3 in the tumor microenvironment enhances adoptive T cell therapy

Abstract Adoptive T cell transfer has shown promise in several malignancies. However, the need to expand antigen-specific T cells for prolonged periods ex vivo and the immunosuppressive effects of the tumor microenvironment severely limit its antitumor efficacy. To overcome these challenges, we tested the feasibility of stimulating and expanding transferred T cells in vivo by targeting Stat3, an oncogenic transcription factor critical for mediating tumor immunosuppression. In contrast to Stat3+/+ CD8+ T cells, transferred naïve CD8+ T cells with genetic Stat3 inhibition, effectively infiltrated tumors where they proliferated robustly, resulting in increased tumor antigen-specific CD8+ T cell activity and tumor growth inhibition. To facilitate the clinical translation of these findings, we further tested sunitinib, an FDA-approved tyrosine kinase inhibitor that has been shown to block Stat3 in several cell types. Sunitinib treatment combined with adoptive T cell transfer of Stat3−/− CD8+ T cells resulted in significant tumor growth inhibition in both immunosuppressed and immunocompetent hosts. Furthermore, sunitinib enhanced tumor T cell infiltration, dendritic cell activation and decreased tumor conversion of T regulatory cells. Targeting Stat3 in tumor cells and immune cells with sunitinib represents a novel approach to overcoming the obstacles that currently face adoptive T cell transfer therapy, which can be translated for immediate use in the clinic for treating cancer. Supported by R01CA146092-01, K12 CA 01727, Kure It Kidney Cancer Research Fund and the Richard and Nancy Bloch Research Fund. 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 5604.

Abstract 2602: The cardiotoxicity and myocyte damage caused by small molecule anticancer tyrosine kinase inhibitors is correlated with lack of target specificity

Abstract The use of the new anticancer tyrosine kinase inhibitors (TKI) has revolutionized the treatment of certain cancers. However, the use of some of these results in cardiotoxicity. Large scale profiling data recently made available for the binding of 7 of the 9 FDA-approved tyrosine kinase inhibitors to a panel of 317 kinases has allowed us to correlate kinase inhibitor binding selectivity scores with TKI-induced damage to neonatal rat cardiac myocytes. The 7 TKIs that were the subject of this study were: imatinib (Gleevec), sunitinib (Sutent), dasatinib (Sprycel), sorafenib (Nexavar), lapatinib (Tykerb), gefitinib (Iressa), and erlotinib (Tarceva). The tyrosine kinase selectivity scores, but not the serine-threonine kinase scores, were highly correlated with the myocyte damaging effects of the TKIs. Additionally, we showed that damage to myocytes gave a good rank order correlation with clinical cardiotoxicity. Finally, strength of TKI binding to colony stimulating factor 1 receptor (CSF1R) was highly correlated with myocyte damage, thus possibly implicating this kinase in contributing to TKI-induced cardiotoxicity. Support: CIHR and a Canada Research Chair in Drug Development to BBH. 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 2602.