Lauren Moran

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.

Crizotinib-Resistant Mutants of EML4-ALK Identified Through an Accelerated Mutagenesis Screen

Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non‐small‐cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non‐small‐cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib‐resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK‐positive non‐small‐cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib’s narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance.

Discovery of Brigatinib (AP26113), a Phosphine Oxide-Containing, Potent, Orally Active Inhibitor of Anaplastic Lymphoma Kinase

In the treatment of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase positive (ALK+) non-small-cell lung cancer (NSCLC), secondary mutations within the ALK kinase domain have emerged as a major resistance mechanism to both first- and second-generation ALK inhibitors. This report describes the design and synthesis of a series of 2,4-diarylaminopyrimidine-based potent and selective ALK inhibitors culminating in identification of the investigational clinical candidate brigatinib. A unique structural feature of brigatinib is a phosphine oxide, an overlooked but novel hydrogen-bond acceptor that drives potency and selectivity in addition to favorable ADME properties. Brigatinib displayed low nanomolar IC50s against native ALK and all tested clinically relevant ALK mutants in both enzyme-based biochemical and cell-based viability assays and demonstrated efficacy in multiple ALK+ xenografts in mice, including Karpas-299 (anaplastic large-cell lymphomas [ALCL]) and H3122 (NSCLC). Brigatinib represents the most clinically advanced phosphine oxide-containing drug candidate to date and is currently being evaluated in a global phase 2 registration trial.

The Potent ALK Inhibitor Brigatinib (AP26113) Overcomes Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in Preclinical Models

Purpose: Non–small cell lung cancers (NSCLCs) harboring ALK gene rearrangements (ALK+) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib. Experimental Design: A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and in vivo activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib–ALK co-structure was determined. Results: Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK+ cell lines, brigatinib inhibited native ALK (IC50, 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK+ tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses. Conclusions: Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK+, crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. Clin Cancer Res; 22(22); 5527–38. ©2016 AACR.

Antitumor Activity of Ridaforolimus and Potential Cell-Cycle Determinants of Sensitivity in Sarcoma and Endometrial Cancer Models

Ridaforolimus is a nonprodrug rapamycin analogue that potently inhibits mTOR and has shown significant activity in patients with metastatic sarcoma and endometrial cancer, two diseases where high unmet need remains. Here, we evaluated the activity of ridaforolimus in preclinical models of these tumor types and used these models to explore molecular correlates of sensitivity. The in vitro sensitivity of a panel of sarcoma and endometrial cancer cell lines was established by measuring the effect of ridaforolimus on cell proliferation rate, revealing broad inhibition at low nanomolar concentrations. Additional benefit was found when ridaforolimus was combined with agents used to treat sarcoma and endometrial cancer patients. In vivo, potent antitumor activity of ridaforolimus associated with inhibition of mTOR signaling was observed in sarcoma and endometrial xenograft models. Immunoblot analysis was conducted to assess the expression and activation state of multiple signaling proteins in the phosphoinositide-3-kinase/AKT/mTOR and cell-cycle pathways. In endometrial but not sarcoma cell lines, the absence of PTEN or elevated levels of phosphorylated or total AKT was associated with greater sensitivity. However, in both tumor types, the proportion of cells in the G0–G1 phase before treatment correlated significantly with ridaforolimus sensitivity. Consistent with this, expression of several G1 phase cell-cycle proteins, notably p21 and p27, was higher in more sensitive lines. These results underscore the promise of ridaforolimus as a single agent or combination treatment of these tumor types and suggest novel potential predictive biomarkers of sensitivity to an mTOR inhibitor based on cell-cycle status. Mol Cancer Ther; 10(10); 1959–68. ©2011 AACR.

Abstract LB-298: AP26113, a potent ALK inhibitor, overcomes mutations in EML4-ALK that confer resistance to PF-02341066 (PF1066)

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC AP26113 is a potent and selective inhibitor of anaplastic lymphoma kinase (ALK) (AACR 2010; #3623). Activating gene rearrangements of ALK, such as EML4-ALK, have been identified as driver mutations in NSCLC and other cancers. There is strong precedence for the development of resistance to targeted therapies that inhibit driver mutations. Kinase domain mutations that confer resistance in patients have been successfully predicted by in vitro mutagenesis screens in BaF3 cells (e.g. BCR-ABL in CML). Here, the BaF3 system was used to identify mutations in ALK that confer resistance to PF1066, a clinically validated dual Met/ALK inhibitor (ASCO 2009; #3509), or AP26113. PF1066-resistant mutations were identified at all concentrations tested (up to 2000 nM). In contrast, 1000 nM AP26113 completely suppressed emergence of resistance. Six mutations, all in the kinase domain, were identified that confer some degree of resistance to 1 or both compounds (Table). AP26113 inhibited viability of BaF3 cells expressing these mutants with IC50s of 23 - 269 nM. PF1066 inhibited viability with IC50s of 311 -1419 nM, with 3 mutants having sensitivity indistinguishable from parental BaF3 cells, which lack EML4-ALK. The 2 mutations that confer the greatest resistance to PF1066 were examined in a BaF3 xenograft model in which compounds were administered daily by oral dosing. A 200 mg/kg dose of PF1066 induced regression of tumors expressing native EML4-ALK but was completely inactive against G1269S or L1196M (gatekeeper) mutants. In contrast, AP26113 induced regression of tumors expressing native EML4-ALK and the G1269S and L1196M mutants at 25, 50 and 50 mg/kg, respectively. Analysis of ALK phosphorylation in tumors demonstrated strong inhibition of the mutants by 50 mg/kg AP26113 but not 200 mg/kg PF1066. These results identify several mutations that may confer resistance to PF1066 in patients and suggest that more potent compounds such as AP26113 may be required to overcome such resistance. View this table: Sensitivity of BaF3 cells expressing native and mutant EML4-ALK to PF-02341066 and AP26113 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 LB-298.

Abstract 1794: AP26113 is a dual ALK/EGFR inhibitor: Characterization against EGFR T790M in cell and mouse models of NSCLC

Background: The EGFR T790M gatekeeper mutation accounts for ∼50% of resistance observed in patients treated with first generation inhibitors that target activated variants of EGFR. Multiple irreversible T790M inhibitors are in development but can exhibit skin and GI toxicity due to co-inhibition of native (endogenous) EGFR, suggesting that T790M-selective agents will be required. Previously, we have identified AP26113 as a potent ALK inhibitor that maintains activity against crizotinib-resistant variants, including the L1196M gatekeeper mutant. Here we further characterize AP261139s activity as a reversible inhibitor of activated and T790M-mutant EGFR. Methods: The activity of AP26113 against native EGFR, or activated forms (delE746_A750 [DEL]) with or without a T790M resistance mutation, was examined in NSCLC as well as engineered Ba/F3 cell lines. EGFR activity was assessed by measuring levels of phosphorylated EGFR, in vitro proliferation measured by MTS assay, and in vivo tumor growth measured in mouse xenografts following daily oral dosing. Results: AP26113 did not inhibit native EGFR phosphorylation in a NSCLC cell line (H358) or in engineered Ba/F3 cells (IC50s >3000 nM). In contrast, potent activity was observed against activated forms of EGFR, with or without the T790M mutation. In Ba/F3 cells expressing EGFR-DEL, AP26113 inhibited EGFR phosphorylation and viability with IC50s of 75 and 114 nM, respectively. In Ba/F3 cells expressing EGFR-DEL/T790M, AP26113 inhibited EGFR phosphorylation and viability with IC50s of 15 and 281 nM, respectively. In a NSCLC line expressing EGFR-DEL (HCC827), AP26113 inhibited EGFR phosphorylation with an IC50 of 62 nM and cell growth with a GI50 of 165 nM. In HCC827 cells expressing EGFR-DEL/T790M AP26113 inhibited EGFR phosphorylation with an IC50 of 59 nM and cell growth with a GI50 of 245 nM. AP26113 also exhibited similar potency against HCC827 cells expressing either EGFR-DEL or EGFR-DEL/T790M in a xenograft model, with daily oral doses of 25 mg/kg or greater leading to tumor regression in both models. Anti-tumor activity that was associated with inhibition of EGFR phosphorylation was also seen in a Ba/F3 EGFR-DEL/T790M tumor model. Conclusions: AP26113 is a potent, reversible inhibitor of activated and T790M-mutant EGFR that does not inhibit the native enzyme. Importantly, orally efficacious doses in mice against activated and T790M-mutant EGFR are similar to those active against crizotinib-resistant ALK variants, suggesting that AP26113 is a dual ALK/mutant EGFR inhibitor with potential to target these two well-defined and important subsets of NSCLC. Based on these data, we recently initiated a Phase 1/2 clinical trial of AP26113 (NCT01449461, www.clinicaltrials.gov) in ALK and EGFR+ NSCLC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1794. doi:1538-7445.AM2012-1794

Abstract 3623: Efficacy and pharmacodynamic analysis of AP26113, a potent and selective orally active inhibitor of Anaplastic Lymphoma Kinase (ALK)

Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified in anaplastic large cell lymphoma (ALCL; NPM-ALK) and non-small cell lung cancer (NSCLC; EML4-ALK). The dual Met/ALK inhibitor PF-02341066 (PF1066) has demonstrated promising clinical activity against tumors carrying activating ALK gene rearrangements (Kwak ASCO 2009: #3509) validating ALK as a therapeutic target. Previously, AP26113 was identified as a novel, potent, orally bioavailable ALK inhibitor with demonstrated selectivity over related receptor tyrosine kinase family members IGF-1R and InsR and no inhibition of Met. Here the efficacy and exposure/activity relationship of AP26113 was further characterized in preclinical models and compared to that of PF1066. In a panel of 7 EML4-ALK or NPM-ALK positive NSCLC and ALCL cell lines, the concentration of AP26113 that inhibited growth by 50% (GI50) ranged from 4.2 - 30.8 nM. In each cell line the GI50 for PF1066 was ∼10-fold greater (range 62 - 309 nM). In 4 cells lines tested, the IC50 for inhibition of ALK phosphorylation tracked with potency in cell proliferation assays and was 10-fold greater for PF1066 than AP26113. Across 3 ALK-negative NSCLC and ALCL cell lines the GI50s for AP26113 (503 - 2387 nM) and PF1066 (928 - 1773 nM) were similar. Overall, AP26113 exhibited ∼100-fold selectivity for ALK-positive lines compared with a ∼10-fold selectivity for PF1066. The in vivo activities of daily oral dosing of AP26113 (10, 25 and 50 mg/kg) and PF1066 (25, 50 and 100 mg/kg) were examined in Karpas-299 ALCL (2 week dosing) and H3122 NSCLC (3 week dosing) xenograft models. At the highest doses tested, strong regressions were achieved with AP26113, but not PF1066. Tumor growth inhibition by 25 mg/kg and 10 mg/kg doses of AP26113 in the ALCL and NSCLC models, respectively, was similar to that of 100 mg/kg PF1066. In a PK/PD study in the ALCL model, inhibition of ALK phosphorylation after administration of 100 mg/kg PF1066 was intermediate between that observed after administration of 10 or 25 mg/kg AP26113. Results from the analysis of plasma levels of each drug showed that AP26113 had equivalent efficacy to PF1066 at 4- to 10-fold lower levels of exposure (AUC and 24 h trough plasma levels). AP26113 demonstrated favorable properties including moderate in vitro plasma protein binding (≤77% in mouse, rat, monkey, and human plasma), negligible inhibition of major CYP isoforms (IC50 > 10 μM for 3A4, 2C9, 2D6), and good oral bioavailability (multiple animal species). In animal models, AP26113 was well-tolerated at and above predicted clinically effective plasma levels. In conclusion, these data demonstrate that AP26113 is a highly potent and selective inhibitor of ALK and support the clinical evaluation of AP26113 in patients with ALK-driven tumors. 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 3623.

Abstract 5655: AP26113 possesses pan-inhibitory activity versus crizotinib-resistant ALK mutants and oncogenic ROS1 fusions.

AP26113 is a potent, reversible inhibitor of ALK fusions and mutant (but not native) EGFR. To overcome mutation-based resistance, AP26113 was designed to maintain activity against crizotinib-resistant ALK variants such as the gatekeeper ALK mutant L1196M. In an ongoing phase 1 dose-escalation study, AP26113 has achieved steady-state trough concentrations in excess of 1 μM and demonstrated promising clinical activity in both crizotinib-resistant and naive ALK-positive NSCLC patients and preliminary evidence of activity in patients with mutant EGFR (#439O, ESMO 2012). To further assess the activity of AP26113 against crizotinib-resistant ALK mutants, and the structurally related ROS1 fusions recently identified in NSCLC, we engineered Ba/F3 cells to express the appropriate oncogenic drivers and evaluated their sensitivities to crizotinib and AP26113. Ba/F3 cell lines expressing clinically-identified EML4-ALK mutants (T1151T insertion, S1206Y, D1203N, L1196M, G1202R, F1174C and C1156Y) had substantially reduced sensitivity to crizotinib (viability IC50= 363-1296 nM) compared with native EML4-ALK (IC50= 137 nM). These data are consistent with the identification of these mutants in patients with acquired crizotinib resistance. AP26113 potently inhibited both native (IC50= 21 nM) and crizotinib-resistant ALK mutants (IC50= 26-254 nM) at concentrations substantially below the clinically achievable trough levels of AP26113 (1 μM), suggesting it may possess a pan-ALK inhibitory profile. AP26113 also effectively inhibited the viability of Ba/F3 cells expressing CD74-ROS1 (IC50= 18 nM), FIG-ROS1 (IC50= 31 nM), SDC4-ROS1 (IC50= 16 nM) and EZR-ROS1 (IC50= 41 nM) thus demonstrating that it is an equipotent inhibitor of ALK and ROS1. In a Ba/F3 CD74-ROS1 xenograft model, AP26113 inhibited tumor growth in a dose-dependent manner, with 50 mg/kg AP26113 inducing substantial tumor regression. Since ROS1 and ALK are structurally related and similarly sensitive to crizotinib, we reasoned that acquired drug resistance in ROS1-driven tumors may also occur via mutation. We therefore generated Ba/F3 cells driven by L2026M gatekeeper mutant forms of CD74-ROS1 and FIG-ROS1 and tested their drug sensitivity. Interestingly, the inhibitory capacity of AP26113 was unaffected by L2026M. In contrast, crizotinib potencies were reduced approximately 4-fold. In conclusion, we have demonstrated that AP26113 inhibits clinically relevant crizotinib- resistant ALK mutants and oncogenic ROS1 fusions recently identified in NSCLC irrespective of the fusion partner. Importantly, since AP26113 maintains potent activity against mutant forms of ALK and ROS1 at concentrations substantially below its human trough concentrations these data suggest that AP26113 may be able to prevent the emergence of drug-resistant mutants in NSCLC patients. Citation Format: Rachel M. Squillace, Rana Anjum, David Miller, Sadanand Vodala, Lauren Moran, Frank Wang, Tim Clackson, Andrew P. Garner, Victor M. Rivera. AP26113 possesses pan-inhibitory activity versus crizotinib-resistant ALK mutants and oncogenic ROS1 fusions. [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 5655. doi:10.1158/1538-7445.AM2013-5655

Abstract 3560: Ponatinib (AP24534), a potent pan-FGFR inhibitor with activity in multiple FGFR-driven cancer models

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Members of the fibroblast growth factor receptor family of kinases (FGFR1-4) are dysregulated in multiple cancers. Ponatinib is a multi-targeted tyrosine kinase inhibitor (TKI) with potent activity against BCR-ABL being investigated in a pivotal phase 2 trial in patients with chronic myeloid leukemia (CML). Previously, ponatinib has been shown to potently inhibit FGFR1-4 kinase activity. Here the activity of ponatinib is explored against FGFR1-4 activated via multiple discrete mechanisms in a variety of cancer types in vitro and in vivo. Results: The cellular activity of ponatinib was first examined in Ba/F3 cells engineered to express activated FGFRs. Ponatinib selectively inhibited viability of cells expressing FGFR1-4, with IC50s of 8 to 34 nM, while having no effect on viability of parental Ba/F3 cells (IC50 >1000 nM). Likewise, ponatinib inhibited phosphorylation of FGFR1-4 with IC50s of 29 to 39 nM. Four other TKIs in clinical development that have been reported to have anti-FGFR activity were substantially less potent: dovitinib (IC50: 34 to 235 nM), cediranib (54 to >1000 nM), BIBF 1120 (214 to >1000 nM) and brivanib (503 to >1000 nM). Next the activity of ponatinib was examined in a panel of cell lines representing multiple tumor types and containing FGFRs dysregulated by a variety of mechanisms. Ponatinib potently inhibited growth of breast cancer cells containing amplified FGFR1 or FGFR2 (GI50: 14-69 nM) and of gastric cancer cells with amplified FGFR2 (GI50: 10-25 nM). In endometrial cancer cells with an activating mutation in the kinase domain of FGFR2 (N549K), or a mutation that increases ligand binding (S252W), ponatinib inhibited growth with GI50s of 14-61 nM. In bladder cancer cells with a mutation in FGFR3 that causes constitutive dimerization (S249C), ponatinib inhibited growth with GI50s of 103-181 nM. In all cell lines, the effects on cell growth were accompanied by inhibition of FGFR or FRS2α phosphorylation. In comparison, ponatinib was less potent in a panel of cell lines lacking expression of activated FGFRs (GI50: 372 nM to >1 uM). The 4 other TKIs examined were less active compared to ponatinib in all FGFR mutant cell lines examined. Daily oral dosing of ponatinib (30 mg/kg) to mice reduced growth of FGFR2N549K endometrial and FGFR3S249C bladder tumor xenografts by approximately 80% and induced regression of gastric tumors expressing amplified FGFR2 by 50%. In all 3 models, dose-dependent inhibition of FGFR phosphorylation in the tumor was demonstrated. Conclusion: Ponatinib exhibits potent, pan-FGFR inhibitory activity that compares favorably to dovitinib, cediranib, BIBF 1120 and brivanib. These results provide a strong rationale for clinical evaluation of ponatinib in FGFR-driven cancers. 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 3560. doi:10.1158/1538-7445.AM2011-3560