Joseph M. Gozgit

Ponatinib (AP24534), a Multitargeted Pan-FGFR Inhibitor with Activity in Multiple FGFR-Amplified or Mutated Cancer Models

Members of the fibroblast growth factor receptor family of kinases (FGFR1–4) are dysregulated in multiple cancers. Ponatinib (AP24534) is an oral multitargeted tyrosine kinase inhibitor being explored in a pivotal phase II trial in patients with chronic myelogenous leukemia due to its potent activity against BCR-ABL. Ponatinib has also been shown to inhibit the in vitro kinase activity of all four FGFRs, prompting us to examine its potential as an FGFR inhibitor. In Ba/F3 cells engineered to express activated FGFR1–4, ponatinib potently inhibited FGFR-mediated signaling and viability with IC50 values <40 nmol/L, with substantial selectivity over parental Ba/F3 cells. In a panel of 14 cell lines representing multiple tumor types (endometrial, bladder, gastric, breast, lung, and colon) and containing FGFRs dysregulated by a variety of mechanisms, ponatinib inhibited FGFR-mediated signaling with IC50 values <40 nmol/L and inhibited cell growth with GI50 (concentration needed to reduce the growth of treated cells to half that of untreated cells) values of 7 to 181 nmol/L. Daily oral dosing of ponatinib (10–30 mg/kg) to mice reduced tumor growth and inhibited signaling in all three tumor models examined. Importantly, the potency of ponatinib in these models is similar to that previously observed in BCR-ABL–driven models and plasma levels of ponatinib that exceed the IC50 values for FGFR1–4 inhibition can be sustained in patients. These results show that ponatinib is a potent pan-FGFR inhibitor and provide strong rationale for its evaluation in patients with FGFR-driven cancers. Mol Cancer Ther; 11(3); 690–9. ©2012 AACR.

FGFR1 mRNA and Protein Expression, not Gene Copy Number, Predict FGFR TKI Sensitivity across All Lung Cancer Histologies

Purpose: FGFR1 gene copy number (GCN) is being evaluated as a biomarker for FGFR tyrosine kinase inhibitor (TKI) response in squamous cell lung cancers (SCC). The exclusive use of FGFR1 GCN for predicting FGFR TKI sensitivity assumes increased GCN is the only mechanism for biologically relevant increases in FGFR1 signaling. Herein, we tested whether FGFR1 mRNA and protein expression may serve as better biomarkers of FGFR TKI sensitivity in lung cancer. Experimental Design: Histologically diverse lung cancer cell lines were submitted to assays for ponatinib sensitivity, a potent FGFR TKI. A tissue microarray composed of resected lung tumors was submitted to FGFR1 GCN, and mRNA analyses and the results were validated with The Cancer Genome Atlas (TCGA) lung cancer data. Results: Among 58 cell lines, 14 exhibited ponatinib sensitivity (IC50 values ≤ 50 nmol/L) that correlated with FGFR1 mRNA and protein expression, but not with FGFR1 GCN or histology. Moreover, ponatinib sensitivity associated with mRNA expression of the ligands, FGF2 and FGF9. In resected tumors, 22% of adenocarcinomas and 28% of SCCs expressed high FGFR1 mRNA. Importantly, only 46% of SCCs with increased FGFR1 GCN expressed high mRNA. Lung cancer TCGA data validated these findings and unveiled overlap of FGFR1 mRNA positivity with KRAS and PIK3CA mutations. Conclusions: FGFR1 dependency is frequent across various lung cancer histologies, and FGFR1 mRNA may serve as a better biomarker of FGFR TKI response in lung cancer than FGFR1 GCN. The study provides important and timely insight into clinical testing of FGFR TKIs in lung cancer and other solid tumor types. Clin Cancer Res; 20(12); 3299–309. ©2014 AACR.

Potent Activity of Ponatinib (AP24534) in Models of FLT3-Driven Acute Myeloid Leukemia and Other Hematologic Malignancies

Ponatinib (AP24534) is a novel multitargeted kinase inhibitor that potently inhibits native and mutant BCR-ABL at clinically achievable drug levels. Ponatinib also has in vitro inhibitory activity against a discrete set of kinases implicated in the pathogenesis of other hematologic malignancies, including FLT3, KIT, fibroblast growth factor receptor 1 (FGFR1), and platelet derived growth factor receptor α (PDGFRα). Here, using leukemic cell lines containing activated forms of each of these receptors, we show that ponatinib potently inhibits receptor phosphorylation and cellular proliferation with IC50 values comparable to those required for inhibition of BCR-ABL (0.3 to 20 nmol/L). The activity of ponatinib against the FLT3-ITD mutant, found in up to 30% of acute myeloid leukemia (AML) patients, was particularly notable. In MV4-11 (FLT3-ITD+/+) but not RS4;11 (FLT3-ITD−/−) AML cells, ponatinib inhibited FLT3 signaling and induced apoptosis at concentrations of less than 10 nmol/L. In an MV4-11 mouse xenograft model, once daily oral dosing of ponatinib led to a dose-dependent inhibition of signaling and tumor regression. Ponatinib inhibited viability of primary leukemic blasts from a FLT3-ITD positive AML patient (IC50 4 nmol/L) but not those isolated from 3 patients with AML expressing native FLT3. Overall, these results support the investigation of ponatinib in patients with FLT3-ITD–driven AML and other hematologic malignancies driven by KIT, FGFR1, or PDGFRα. Mol Cancer Ther; 10(6); 1028–35. ©2011 AACR.

Ponatinib (AP24534) Is a Novel Potent Inhibitor of Oncogenic RET Mutants Associated With Thyroid Cancer

CONTEXT The RET tyrosine kinase encoding gene acts as a dominantly transforming oncogene in thyroid carcinoma and other malignancies. Ponatinib (AP24534) is an oral ATP-competitive tyrosine kinase inhibitor that is in advanced clinical experimentation in leukemia. OBJECTIVE We tested whether ponatinib inhibited RET kinase and oncogenic activity. METHODS Ponatinib activity was studied by an in vitro RET immunocomplex kinase assay and immunoblotting. The effects of ponatinib on proliferation of human TT, MZ-CRC-1, and TPC-1 thyroid carcinoma cells, which harbor endogenous oncogenic RET alleles, and of NIH3T3 fibroblasts transfected with oncogenic RET mutants were determined. Ponatinib activity on TT cell xenografted tumors in athymic mice was measured. RESULTS Ponatinib inhibited immunopurified RET kinase at the IC₅₀ of 25.8 nM (95% confidence interval [CI] = 23.15-28.77 nM). It also inhibited (IC₅₀ = 33.9 nM; 95% CI = 26.41-43.58 nM) kinase activity of RET/V804M, a RET mutant displaying resistance to other tyrosine kinase inhibitor. Ponatinib blunted phosphorylation of point-mutant and rearranged RET-derived oncoproteins and inhibited proliferation of RET-transformed fibroblasts and RET mutant thyroid carcinoma cells. Finally, after 3 weeks of treatment with ponatinib (30 mg/kg/d), the volume of TT cell (medullary thyroid carcinoma) xenografts was reduced from 133 mm³ to an unmeasurable size (difference = 133 mm³, 95% CI = -83 to 349 mm³) (P < .001). Ponatinib-treated TT cell tumors displayed a reduction in the mitotic index, RET phosphorylation, and signaling. CONCLUSIONS Ponatinib is a potent inhibitor of RET kinase and has promising preclinical activity in models of RET-driven medullary thyroid carcinoma.

Ponatinib inhibits polyclonal drug-resistant KIT oncoproteins and shows therapeutic potential in heavily pretreated gastrointestinal stromal tumor (GIST) patients.

Purpose: KIT is the major oncogenic driver of gastrointestinal stromal tumors (GIST). Imatinib, sunitinib, and regorafenib are approved therapies; however, efficacy is often limited by the acquisition of polyclonal secondary resistance mutations in KIT, with those located in the activation (A) loop (exons 17/18) being particularly problematic. Here, we explore the KIT-inhibitory activity of ponatinib in preclinical models and describe initial characterization of its activity in patients with GIST. Experimental Design: The cellular and in vivo activities of ponatinib, imatinib, sunitinib, and regorafenib against mutant KIT were evaluated using an accelerated mutagenesis assay and a panel of engineered and GIST-derived cell lines. The ponatinib–KIT costructure was also determined. The clinical activity of ponatinib was examined in three patients with GIST previously treated with all three FDA-approved agents. Results: In engineered and GIST-derived cell lines, ponatinib potently inhibited KIT exon 11 primary mutants and a range of secondary mutants, including those within the A-loop. Ponatinib also induced regression in engineered and GIST-derived tumor models containing these secondary mutations. In a mutagenesis screen, 40 nmol/L ponatinib was sufficient to suppress outgrowth of all secondary mutants except V654A, which was suppressed at 80 nmol/L. This inhibitory profile could be rationalized on the basis of structural analyses. Ponatinib (30 mg daily) displayed encouraging clinical activity in two of three patients with GIST. Conclusion:Ponatinib possesses potent activity against most major clinically relevant KIT mutants and has demonstrated preliminary evidence of activity in patients with refractory GIST. These data strongly support further evaluation of ponatinib in patients with GIST. Clin Cancer Res; 20(22); 5745–55. ©2014 AACR.

Combined targeting of FGFR2 and mTOR by ponatinib and ridaforolimus results in synergistic antitumor activity in FGFR2 mutant endometrial cancer models

PurposeActivating mutations in FGFR2 have been identified as potential therapeutic targets in endometrial cancer, typically occurring alongside genetic alterations that disrupt the mTOR pathway, such as PTEN loss. These observations suggest that the mTOR pathway may act in concert with oncogenic FGFR2 to drive endometrial cancer growth in a subset of patients. The aim of this study was to examine the therapeutic potential of a rational drug combination based on the simultaneous targeting of mutant-FGFR2 and mTOR-driven signaling pathways in endometrial cancer cells.MethodsPonatinib is an oral multitargeted kinase inhibitor that potently inhibits all 4 members of the FGFR family. Ridaforolimus is a selective inhibitor of mTOR that has demonstrated positive clinical activity in endometrial cancer. The combinatorial effects of ponatinib and ridaforolimus on growth of endometrial cancer models, and their modes of action, were evaluated in vitro and in vivo.ResultsThe combination of ponatinib and ridaforolimus had a synergistic effect on the in vitro growth of endometrial lines bearing an activating FGFR2 mutation, irrespective of PTEN status. Concomitant inhibition of both FGFR2 and mTOR signaling pathways was observed, with simultaneous blockade resulting in enhanced cell cycle arrest. Ponatinib and ridaforolimus each demonstrated inhibition of tumor growth in vivo, but dual inhibition by the combination of agents resulted in superior efficacy and induced tumor regression in an endometrial xenograft.ConclusionsThese encouraging preclinical findings suggest the inhibition of both FGFR2 and mTOR by the ponatinib–ridaforolimus combination may provide a new therapeutic strategy to treat advanced endometrial cancers with dual pathway dysregulation.

Ponatinib in patients with refractory acute myeloid leukaemia: Findings from a phase 1 study

Activating mutations in the FMS-like tyrosine kinase-3 (FLT3), a tyrosine kinase receptor important in haematopoiesis, are among the most common molecular aberrations in acute myeloid leukaemia (AML), occurring in 30% of adult patients (Levis & Small 2003). Common FLT3-activating mutations include FLT3 internal tandem duplications (FLT3-ITDs), detected in about 23% of AML patients, and point mutations within the tyrosine kinase domain, found in about 8% (Levis & Small 2003). These mutations result in a constitutively active FLT3 receptor, leading to growth factor–independent proliferation and survival of leukaemic cells and conferring poor prognosis (Levis & Small 2003). Clinical studies of single-agent first-generation FLT3 inhibitors have demonstrated clinical activity, with responses that are typically short-lived and mostly partial or complete responses with incomplete haematopoietic recovery. This may be due to suboptimal potency and/or pharmacokinetics, leading to insufficient or transient target inhibition, or concomitant c-kit inhibition (Knapper 2011). Recently, high potency second-generation FLT3 inhibitors (eg, quizartinib) have shown substantial efficacy as monotherapy, suggesting a potency threshold for clinical benefit (Knapper 2011). The validation of FLT3-ITD as a therapeutic target has rekindled interest in developing and testing new potent FLT3 inhibitors in AML patients with FLT3-ITD mutations (Smith et al, 2012). Ponatinib is a novel, orally administered tyrosine kinase inhibitor (TKI) and a potent pan–BCR-ABL1 inhibitor (O’Hare et al, 2009). Based on results in patients with chronic myeloid leukaemia (CML) and Philadelphia chromosome–positive acute lymphoblastic leukaemia (Ph+ ALL) in phase 1 and phase 2 clinical trials (Cortes et al 2012a, Cortes et al (2012b), ponatinib (45 mg once daily) has been approved in the United States for the treatment of patients with CML and Ph+ ALL that is resistant or intolerant to prior TKI therapy. Preclinical studies revealed that ponatinib also potently inhibits FLT3, leading to apoptosis of leukaemic cell lines carrying the FLT3-ITD mutation and tumour regression in xenograft models, suggesting the potential for activity in patients with AML (Gozgit et al, 2011). Additionally, ponatinib appears to retain activity against the clinically-relevant quizartinib-resistant mutant FLT3-ITD F691L (Smith et al, 2013). Here we report the first clinical experience with ponatinib in 12 AML patients included in the phase 1 study. Methods are described in the on-line supporting information. The median age of these patients was 49 (30-72) years. The median time from diagnosis to treatment was 1 year. Patients received a median of 3 (1-7) prior therapies; 58% had received 3 or more prior therapies (Table I and Table S1). Mutational analysis in a central laboratory confirmed the presence of FLT3-ITD in 7 patients (58%). Three additional patients did not have an adequate DNA sample at study entry; however, they had a history of FLT3-ITD mutation—as reported by the investigator—and they are included in the FLT3-ITD mutation– positive group for these analyses. Three patients (all FLT3-ITD mutation positive) were previously treated with one or more FLT3 inhibitors (sorafenib, quizartinib, and/or IMC-EB10); one patient progressed on IMC-EB10 and had a partial response to sorafenib, one patient had a complete response to sorafenib and a partial response to quizartinib, and one patient had a partial response to quizartinib. Seven patients (70%) with FLT3-ITD mutation were FLT3 inhibitor– naive (Table I). The median treatment duration was 52 (10-173) days. At the time of analysis, all patients had discontinued ponatinib: 5 (42%) due to death (all unrelated to ponatinib), 3 (25%) due to adverse events (AEs: unrelated central nervous system [CNS] haemorrhage, possibly related acute pancreatitis, unrelated graft vs host disease), 2 (17%) due to progressive disease (PD), and 2 (17%) due to investigator decision (Table I). Table 1 Selected baseline characteristics, treatment duration, response, and reasons for discontinuation by individual patients with AML Nine patients experienced at least one treatment-related AE. The most common treatment-related AEs occurring in 2 or more patients were pancreatitis (n=3) and petechiae (n=2). Three patients experienced a treatment-related serious AE (SAE) of pancreatitis (all grade 2), which was a dose-limiting toxicity in this trial (Cortes et al, 2012a). Pancreatitis resolved in 2 patients after dose interruption, lasting 3 days in one patient and 8 days in the other. These 2 patients continued therapy at a reduced dose (30 mg) and were subsequently re-escalated to 45 mg without recurrence. The third patient discontinued therapy per investigator decision. Additional details regarding treatment-emergent AEs and SAEs can be found in Table S2. Seven patients died during the study for reasons not related to ponatinib: disease progression (n=3), multiorgan failure (n=2), pneumonia and sepsis (n=1), and CNS haemorrhage (n=1) (Table I). Ponatinib had an acceptable safety profile in this small group of patients with refractory AML, similar to that observed in patients with CML and Ph+ ALL. Few treatment-related AEs were reported; the most common was pancreatitis, which was manageable, and re-challenge with ponatinib was possible in most cases. The geometric mean maximal concentration and area under the curve of single-dose ponatinib at day 1, cycle 1 in AML patients were 97 nM and 1441 nM*h, respectively, similar to findings across all 31 patients receiving 45 mg ponatinib (98.8 nM and 1360.1 nM*h). The overall response rate (RR, partial remission or better) was 3/12 (25%): 2 patients achieved complete remission with incomplete blood count recovery and one patient experienced partial remission (Table I, Fig 1). These 3 responders carried FLT3-ITD mutations and were all FLT3 inhibitor–naive; the duration of ponatinib treatment in these patients was 3 to 6 months. Among 10 patients with FLT3-ITD mutations, RR was 3/10 (30%). Among 7 patients with FLT3-ITD mutations who were FLT3 inhibitor–naive, RR was 3/7 (43%). Three patients (2 FLT3-ITD negative) had stable disease, as they did not meet criteria for complete/partial remission or PD; however, peripheral blood blasts in 2 of these patients decreased considerably (~60-90%) during the first treatment cycle. The RR reported with quizartinib in phase 1 testing was 30% (Cortes et al, 2009) and 10% with sorafenib (Borthakur et al, 2011). Although the sample size reported here is small, these results suggest that ponatinib has clinical activity in AML patients with FLT3-ITD, requiring confirmation in a larger cohort of patients and with additional focus on optimization of response (eg, combination therapy) and response durability. Figure 1 Course of the disease in 3 responders during ponatinib treatment

Nonamplified FGFR1 Is a Growth Driver in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is associated with asbestos exposure and is a cancer that has not been significantly affected by small molecule-based targeted therapeutics. Previously, we demonstrated the existence of functional subsets of lung cancer and head and neck squamous cell carcinoma (HNSCC) cell lines in which fibroblast growth factor receptor (FGFR) autocrine signaling functions as a nonmutated growth pathway. In a panel of pleural mesothelioma cell lines, FGFR1 and FGF2 were coexpressed in three of seven cell lines and were significantly associated with sensitivity to the FGFR-active tyrosine kinase inhibitor (TKI), ponatinib, both in vitro and in vivo using orthotopically propagated xenografts. Furthermore, RNAi-mediated silencing confirmed the requirement for FGFR1 in specific mesothelioma cells and sensitivity to the FGF ligand trap, FP-1039, validated the requirement for autocrine FGFs. None of the FGFR1-dependent mesothelioma cells exhibited increased FGFR1 gene copy number, based on a FISH assay, indicating that increased FGFR1 transcript and protein expression were not mediated by gene amplification. Elevated FGFR1 mRNA was detected in a subset of primary MPM clinical specimens and like MPM cells; none harbored increased FGFR1 gene copy number. These results indicate that autocrine signaling through FGFR1 represents a targetable therapeutic pathway in MPM and that biomarkers distinct from increased FGFR1 gene copy number such as FGFR1 mRNA would be required to identify patients with MPM bearing tumors driven by FGFR1 activity. Implications: FGFR1 is a viable therapeutic target in a subset of MPMs, but FGFR TKI-responsive tumors will need to be selected by a biomarker distinct from increased FGFR1 gene copy number, possibly FGFR1 mRNA or protein levels. Mol Cancer Res; 12(10); 1460–9. ©2014 AACR.

Validation of a Targeted RNA Sequencing Assay for Kinase Fusion Detection in Solid Tumors

Kinase gene fusions are important drivers of oncogenic transformation and can be inhibited with targeted therapies. Clinical grade diagnostics using RNA sequencing to detect gene rearrangements in solid tumors are limited, and the few that are available require prior knowledge of fusion break points. To address this, we have analytically validated a targeted RNA sequencing assay (OSU-SpARKFuse) for fusion detection that interrogates complete transcripts from 93 kinase and transcription factor genes. From a total of 74 positive and 36 negative control samples, OSU-SpARKFuse had 93.3% sensitivity and 100% specificity for fusion detection. Assessment of repeatability and reproducibility revealed 96.3% and 94.4% concordance between intrarun and interrun technical replicates, respectively. Application of this assay on prospective patient samples uncovered OLFM4 as a novel RET fusion partner in a small-bowel cancer and led to the discovery of a KLK2-FGFR2 fusion in a patient with prostate cancer who subsequently underwent treatment with a pan-fibroblast growth factor receptor inhibitor. Beyond fusion detection, OSU-SpARKFuse has built-in capabilities for discovery research, including gene expression analysis, detection of single-nucleotide variants, and identification of alternative splicing events.

Abstract 2084: Ponatinib is a highly potent inhibitor of activated variants of RET found in MTC and NSCLC .

Background: RET kinase is dysregulated by activating mutations or fusion gene formation in multiple cancers, including medullary thyroid cancer (MTC) and non-small cell lung cancer (NSCLC). RET mutations are found in 50-95% of MTCs, with mutations at C634R (extracellular domain) and M918T (kinase domain) predominating. Recently, a translocation that results in formation of a KIF5B-RET fusion gene has been identified in 1-2% of NSCLCs. Ponatinib (AP24534) is a multi-targeted tyrosine kinase inhibitor (TKI) with potent activity against BCR-ABL being investigated in patients with chronic myeloid leukemia. Previously, ponatinib has been shown to potently inhibit the in vitro kinase activity of RET and the viability of Ba/F3 cells transformed to IL-3 independence through expression of a RET kinase domain artificially-activated via fusion to the TEL dimerization domain. Here, the activity of ponatinib was examined in Ba/F3 cells transformed with the most common, naturally-occurring activated variants of RET found in MTC and NSCLC. In addition, the activity of ponatinib was compared to that of 4 other multi-targeted TKIs with anti-RET activity in clinical development: vandetanib, cabozantinib, sunitinib and sorafenib. Results: Ponatinib potently inhibited viability of Ba/F3 cells expressing RETC634R, RETM918T and KIF5B-RET, with IC50s of 2, 3, and 11 nM, respectively. Consistent with these effects being due to inhibition of RET, ponatinib inhibited RET phosphorylation with similar potency in each respective cell line (IC50s of 4, 2, and 9 nM). The TKIs vandetanib (IC50: 448, 357 and 773 nM, respectively), cabozantinib (226, 100 and 292 nM), sunitinib (299, 312 and 570 nM) and sorafenib (>1000, 372 and 861 nM) also inhibited viability of Ba/F3 cells expressing RETC634R, RETM918T and KIF5B-RET, however the potency of all 4 TKIs was substantially reduced compared to that of ponatinib. Importantly, trough ponatinib concentrations (64 nM) observed in patients treated once daily with ponatinib (45 mg) substantially exceed IC50s for inhibition of each of the mutationally-activated RET variants. Additional studies to further evaluate the anti-RET activity of ponatinib and other TKIs, including their susceptibility to mutation-based resistance, are ongoing and will be presented. Conclusion: Ponatinib is a highly potent inhibitor of activated variants of RET found in MTC and NSCLC. The potency of ponatinib substantially exceeds that of other TKIs with anti-RET activity that are being evaluated in clinical trials. These results provide strong support for the clinical evaluation of ponatinib in patients with RET-driven cancers. Citation Format: Joseph M. Gozgit, Tzu-Hsiu Chen, Tim Clackson, Victor Rivera. Ponatinib is a highly potent inhibitor of activated variants of RET found in MTC and NSCLC . [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 2084. doi:10.1158/1538-7445.AM2013-2084