Duration Of Target Inhibition Research Articles

Abstract 1972: Preclinical activity of the type II RAF inhibitor tovorafenib in tumor models harboring either a BRAF fusion or a NF1-LOF mutation

Abstract Genomic alterations and dysregulation of the MAPK pathway have been described in many different types of cancers. BRAF V600 point mutations and BRAF fusions, which are found in both pediatric and adult cancers, are oncogenic drivers that drive constitutive activation of the RAF pathway (Jones 2009, Yaeger 2019). Neurofibromatosis 1 (NF1) loss-of-function (LOF) mutations, which occur in many cancer types, result in decreased neurofibromin GAP function, thus activating RAS (Basu 1992). Tovorafenib is an investigational, selective, CNS-penetrant, small molecule type II RAF inhibitor which inhibits both RAF monomers and dimers and has been shown preclinically to impact the KIAA1549::BRAF fusion (Sun 2017). In this study, the impact of tovorafenib alone or in combination with MEK inhibitor, pimasertib, was explored in adult or pediatric tumor models harboring BRAF fusions or NF1-LOF mutations. At clinically relevant doses, tovorafenib treatment resulted in tumor regression in an AGK::BRAF fusion melanoma patient-derived xenograft (PDX) model in vivo. In contrast, little anti-tumor activity was observed in response to tovorafenib in vivo in two tumor models harboring NF1-LOF mutations. Ongoing PK-PD studies in both the AGK::BRAF fusion model and the NF1-LOF models will assess the extent and duration of target inhibition. In vitro, anti-proliferative activity of tovorafenib was also evaluated in NF1-LOF mutant tumor cell lines or PDX models of different tumor types, including melanoma, lung and malignant peripheral nerve sheath tumor (MPNST). While tovorafenib was potent in the BRAF V600E tumor cell line, little activity was observed in the NF1-LOF tumor cell lines. Modulation of phosphorylated-ERK (pERK) was also assessed. At very low concentrations, modest induction of pERK was observed in the NF1-LOF tumor cell lines, with inhibition at higher concentrations. In vitro combination studies were conducted using pimasertib and tovorafenib, or a tool compound. Synergy was observed in a NF1-LOF embryonal rhabdomyosarcoma PDX model ex vivo and a NF1-LOF MPNST cell line in vitro, suggesting that vertical pathway inhibition is impactful in the NF1-LOF mutant setting. Tovorafenib is currently being evaluated as a monotherapy in relapsed or progressive pediatric low-grade glioma (pLGG) harboring RAF alterations (NCT04775485) and in combination with pimasertib in patients ≥12 years of age with recurrent, progressive, or refractory solid tumors harboring MAPK pathway alterations (NCT04985604). Ongoing preclinical translational work will continue to explore potential biomarker-defined tumor indications for these agents. Citation Format: Shubhra Rastogi, Sam Perino, Madhu Lal-Nag, Yujin Wang, Samuel C. Blackman, Eleni Venetsanakos. Preclinical activity of the type II RAF inhibitor tovorafenib in tumor models harboring either a BRAF fusion or a NF1-LOF mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1972.

Abstract 2091: PRN1371, an irreversible, covalent inhibitor of FGFR1-4 exhibits sustained pathway inhibition in cancer cell lines

Abstract Introduction: Multiple human cancers harbor alterations in FGFRs that drive tumor growth, including mutations, translocations and amplifications. PRN1371 is an irreversible, covalent FGFR1-4 inhibitor that exhibits highly selective and sustained inhibition of FGFR which extends well beyond circulating drug concentrations in preclinical models. The duration of inhibition of the FGFR signaling pathway is dependent on protein turnover of FGFR, which may vary depending on the type of FGFR alteration. Thus, we set out to investigate whether the duration of target inhibition differs across cancer cell lines of various lineages harboring different FGFR alterations, including fusions and mutations. Furthermore, as FGFR inhibitors exhibit hyperphosphatemia via on-target pharmacology, we also investigated the duration of target inhibition in primary renal epithelial cells which are wild-type for FGFR. Materials and Methods: Cancer cell lines from several lineages harboring different FGFR alterations were treated with increasing concentrations of PRN1371 in vitro for 1 hour, before washing out the compound. Cells were harvested at various time-points post-washout, protein lysates were generated and assessed for modulation of the downstream signaling pathway by western blot analysis. Results: Dose-dependent inhibition of phospho-ERK was observed in the cancer cell lines tested in response to compound treatment for 1 hour in vitro. Dose dependent partial or full rebound of phospho-ERK back to baseline levels were detected in cancer cell lines after a prolonged period post-washout. In contrast, full rebound of phospho-ERK was observed at 1 hour post washout in response to a non-covalent inhibitor. Studies are on-going to assess duration of pathway inhibition in the primary renal proximal epithelial cells. Conclusions: PRN1371 is a potent, highly selective irreversible inhibitor exhibiting sustained inhibition of FGFR signaling across cancer cell lines harboring different FGFR alterations. The duration of inhibition of the downstream signaling was comparable across cancer cell lines harboring different FGFR alterations, including mutations, fusions and amplification of FGFR and was prolonged when compared to a non-covalent inhibitor. A Phase 1 clinical trial of PRN1371 for the treatment of solid tumors harboring FGFR alterations is ongoing (NCT02608125). Citation Format: Eleni Venetsanakos, Yan Xing, Natalie Loewenstein, J. Michael Bradshaw, Dane Karr, Jacob LaStant, Philip Nunn, Jin Shu, Abha Bommireddi, Jens Oliver Funk, David M. Goldstein, Stefani Wolff, Ken A. Brameld, Steven G. Gourlay. PRN1371, an irreversible, covalent inhibitor of FGFR1-4 exhibits sustained pathway inhibition in cancer cell lines [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 2091. doi:10.1158/1538-7445.AM2017-2091

Abstract 1249: PRN1371, an irreversible, covalent inhibitor of FGFR1, 2, 3 and 4 is highly efficacious in preclinical tumor models

Abstract Introduction: Multiple human cancers harbor alterations in FGFRs that drive tumor growth, including mutations, translocations and amplifications. We developed a covalent, irreversible, highly selective FGFR1, 2, 3 and 4 inhibitor, PRN1371, by targeting a cysteine residue within the kinase domain. This approach enables highly selective and sustained inhibition of FGFR which extends well beyond circulating drug concentrations. PRN1371 is currently in a phase 1 clinical trial for the treatment of solid tumors. Materials and Methods: FGFR1-4 enzymatic activities were measured using the Caliper microfluidics Labchip system. Inhibition of proliferation was assessed over 3 days using Cell-Titer-Glo. SNU16 and RT4 xenograft tumor models were used to investigate pharmacodynamics and efficacy. For tumor inoculation, cancer cells were implanted into the rear flank of immunocompromised mice. Once tumor volume reached a mean average of 175mm3, mice were randomized and treated with FGFR inhibitor. Results: PRN1371 is a novel investigative drug that is a potent ATP competitive, highly selective inhibitor of the FGFR family of protein kinases. PRN1371 binds irreversibly to FGFR1, 2, 3 and 4, leading to long duration of target inhibition. The IC50 values for FGFR1, 2, 3 and 4 are 0.7, 1.3, 4.1 and 19.2 nM, respectively. The IC50 values are 55.0, 12.0, 1.2 and 2.3 nM for FGFR3-V555M, FGFR2-N549H, FGFR3-G697C and FGFR3-K650E, respectively. PRN1371 exhibited no significant activity against 247 other protein kinases. PRN1371 demonstrates potent inhibition of cell proliferation in multiple tumor cell lines driven by dysregulated FGFR signaling. In RT4, RT112, SNU16, AN3-CA, LI7, JHH7, and OPM2 cells, EC50's are 4.0, 4.1, 2.6, 43.3, 33.1, 231 and 14.0 nM, respectively. PRN1371 is rapidly and well-absorbed. Bioavailability is dose and species-dependent. PRN1371 demonstrates potent tumor growth inhibition and regression in FGFR3-fusion expressing RT4 and FGFR2-amplified SNU16 xenograft models, when dosed either continuously, or intermittently, and is generally well-tolerated. PRN1371 is also shown to have significant anti-tumor activity against a panel of PDX tumor xenografts of various lineages harboring different FGFR pathway alterations. The phase 1 clinical trial will evaluate ascending doses of PRN1371 in a conventional “three plus three” design. Expansion cohorts of selected tumor types will then be investigated at the recommended dose level. Conclusion: PRN1371 is a potent, highly selective, irreversible inhibitor exhibiting sustained inhibition of FGFR 1, 2, 3 and 4. A Phase 1 clinical trial of PRN1371 for the treatment of solid tumors is currently ongoing. Citation Format: Eleni Venetsanakos, Michael Bradshaw, David M. Goldstein, Kwan Leung, Dane Karr, Yan Xing, Jacob LaStant, Philip Nunn, Jin Shu, Abha Bommireddi, Steven G. Gourlay, Jens Oliver Funk, Ken A. Brameld. PRN1371, an irreversible, covalent inhibitor of FGFR1, 2, 3 and 4 is highly efficacious in preclinical tumor models. [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 1249.

Abstract C197: Accurins improve the pharmacokinetics, pharmacodynamics, tolerability and anti-tumor activity of the AKT inhibitor MK-2206

Abstract Background: BIND-2206 Accurins are novel polymeric nanoparticles encapsulating MK-2206, a Merck AKT inhibitor in phase 2 trials. MK-2206 targets the phosphatidylinositol 3-kinase (PI3K) pathway via AKT inhibition and has demonstrated therapeutic efficacy in the treatment of cancer albeit with dose limiting toxicities. Accurins have shown promise for targeting oncology agents preferentially to tumors, while limiting systemic exposure. To determine if encapsulation of MK-2206 improves therapeutic index, studies were performed to evaluate pharmacokinetics (PK), pharmacodynamics (PD), tolerability and anti-tumor activity compared to parent MK-2206. Materials and Methods: PK of four Accurin formulations with varying in vitro drug release rates was evaluated. Based on these data, two lead Accurins (BIND-2206C and BIND-2206D) were selected for further characterization. Since AKT inhibitors are documented to induce hyperglycemia in mice, blood glucose levels were also evaluated after acute administration of BIND-2206 Accurins or parent MK-2206 as a measure of tolerability. Using a panel of human tumor xenografts (VCaP, SK-OV-3 and BT-474) anti-tumor activity of the Accurins was assessed in mice. When tumors were established mice were dosed orally with parent MK-2206 or intravenously with BIND-2206 Accurins two or three times per week on a three week cycle. Tumor volume was measured post treatment to determine anti-tumor activity. Following final drug administration, tumors were collected for PK and PD evaluation. Extent and duration of tumor target inhibition was evaluated by measuring pAKT and total AKT using a mesoscale discovery method and was correlated to tumor MK-2206 levels. Results: Accurins BIND-2206C and BIND-2206D significantly enhanced the PK profile of parent MK-2206 by increasing Cmax (33 fold) and AUC (222 fold) and decreasing clearance (38 fold) in mice. In addition, both BIND-2206 Accurins improved tolerability as demonstrated by no hyperglycemic response compared to a 300% increase in blood glucose in nude mice treated with parent MK-2206. In the VCaP human prostate cancer tumor model, BIND-2206C inhibited tumor growth by 88% and BIND-2206D induced 16% tumor regression showing significant enhancement of anti-tumor efficacy compared to 55% tumor growth inhibition achieved by parent MK-2206. This correlated with high tumor exposure at 72 hours and prolonged tumor PD at 96 hours post dose for both Accurins compared to parent MK-2206. A similar increase in tumor exposure and prolonged PD response resulted in enhanced anti-tumor efficacy for BIND-2206 Accurins in the SK-OV-3 human ovarian cancer tumor model. In the BT-474 HER2 over-expressing human breast cancer tumor model, BIND-2206 Accurins did not improve efficacy compared to parent drug, although there was significant and prolonged tumor PD correlating with enhanced tumor PK. This suggests that anti-tumor efficacy is model specific and a combination strategy in the BT-474 model may be advantageous. Conclusions: BIND-2206 Accurins showed differentiated PK, increased tumor exposure, prolonged duration of target inhibition, superior efficacy and enhanced tolerability compared to parent MK-2206. These data suggest that nanoparticle formulations of the AKT inhibitor, MK-2206, may provide improved tolerability and therapeutic efficacy in a clinical setting. Citation Format: Louise Cadzow, Michael H. Lam, Young Ho Song, Maria Figueiredo, Hong Wang, David De Witt, Vincenzo Pucci, Jan-Rung Mo, Eric Lewis-Clark, Heidi Ferguson, Marian Gindy, Susan Low, Steve Zale, Jeff Hrkach, Caroline McGregor, Brian J. Long. Accurins improve the pharmacokinetics, pharmacodynamics, tolerability and anti-tumor activity of the AKT inhibitor MK-2206. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C197.

Abstract 1477: Pharmacodynamic response and anti-tumor activity of the MET inhibitor AZD6094 in papillary renal cell carcinoma patient derived xenograft models

Abstract Papillary renal cell carcinoma (PRCC) is the second most common cancer of the kidney and carries a poor prognosis for patients with non-localized disease. The central role of the hepatocyte growth factor (HGF) receptor MET in PRCC has been explored, demonstrating that MET aberrations, either through mutation, copy number gain, or trisomy of chromosome 7 (the location of MET and HGF) occur in the majority of PRCC cases. We sought to evaluate AZD6094 (HMPL-504), a potent and selective small molecule MET kinase inhibitor, in this disease setting. However, the development of effective therapies targeting MET and other targets in PRCC has been hampered in part by a lack of available preclinical models to test novel targeted therapies. Here we describe for the first time the pharmacodynamic (PD) response and anti-tumor activity of the selective MET inhibitor AZD6094 in two preclinical patient derived xenograft (PDX) models of PRCC (RCC-43b and RCC-47). Both PDX models have increased MET copy number of 8 and 9 copies by FISH in RCC-43b, and RCC-47 respectively, and robust MET protein staining by IHC. AZD6094 treatment resulted in dose dependent anti-tumor responses reaching ∼85% tumor growth inhibition (TGI) when dosed at 2.5 mg/kg daily (qd), stasis when dosed 10 mg/kg qd, and ∼20% regression when dosed at 25 mg/kg qd in the RCC-43b model and ∼63% TGI, ∼89% TGI, ∼64% regression, and ∼96% regression in the RCC-47 model when dosed 0.5, 2.5, 10, and 25 mg/kg qd respectively. The standard of care for RCC, sunitinib, showed no activity in RCC-43b when dosed at 10 mg/kg qd (∼10% TGI, p>0.05 vs vehicle) and ∼60% TGI when dosed at 80 mg/kg qd. Pharmacodynamic analysis of RCC-47 tumors revealed that two hours after an acute dose of AZD6094 pMET levels were reduced >95% at all dose levels tested (0.5 - 25 mg/kg). Eight hours after dosing, pMET levels returned to ∼50% in the 0.5 and 2.5 mg/kg dose groups whereas pMET was still inhibited >90% in the 10 and 25 mg/kg dose groups indicating that the duration of target inhibition was dose related. AZD6094 inhibited multiple signaling nodes including MAPK, PI3K, and EGFR. Finally, at doses that induced tumor regression, AZD6094 resulted in a dose and time dependent induction of cleaved PARP, a marker of cell death. The finding that lower, sub-optimal doses of AZD6094 showed return of pMET 8 hours after a single administration, raised the question whether splitting the dose over a longer duration would increase anti-tumor activity. Indeed, twice a day dosing (bid, 8;16 hours) of AZD6094 at 1.25 mg/kg was more efficacious than daily administration of 2.5 mg/kg resulting in 8% regression compared to 89% TGI (p<0.05). Taken together, these data provide the first report testing therapeutics in preclinical in vivo models of PRCC and support the clinical development of AZD6094 in this indication. Citation Format: Alwin Schuller, Evan Barry, Rhys Jones, Melanie Frigault, Garry Beran, Ryan Henry, David Linsenmayer, Maureen Hattersley, Aaron Smith, Joanne Wilson, Ammar Adam, Michael Zinda, Corinne Reimer, Stephen Fawell, Edwin Clark, Celina D'Cruz. Pharmacodynamic response and anti-tumor activity of the MET inhibitor AZD6094 in papillary renal cell carcinoma patient derived xenograft models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1477. doi:10.1158/1538-7445.AM2015-1477

The MET Inhibitor AZD6094 (Savolitinib, HMPL-504) Induces Regression in Papillary Renal Cell Carcinoma Patient-Derived Xenograft Models.

Papillary renal cell carcinoma (PRCC) is the second most common cancer of the kidney and carries a poor prognosis for patients with nonlocalized disease. The HGF receptor MET plays a central role in PRCC and aberrations, either through mutation, copy number gain, or trisomy of chromosome 7 occurring in the majority of cases. The development of effective therapies in PRCC has been hampered in part by a lack of available preclinical models. We determined the pharmacodynamic and antitumor response of the selective MET inhibitor AZD6094 in two PRCC patient-derived xenograft (PDX) models. Two PRCC PDX models were identified and MET mutation status and copy number determined. Pharmacodynamic and antitumor activity of AZD6094 was tested using a dose response up to 25 mg/kg daily, representing clinically achievable exposures, and compared with the activity of the RCC standard-of-care sunitinib (in RCC43b) or the multikinase inhibitor crizotinib (in RCC47). AZD6094 treatment resulted in tumor regressions, whereas sunitinib or crizotinib resulted in unsustained growth inhibition. Pharmacodynamic analysis of tumors revealed that AZD6094 could robustly suppress pMET and the duration of target inhibition was dose related. AZD6094 inhibited multiple signaling nodes, including MAPK, PI3K, and EGFR. Finally, at doses that induced tumor regression, AZD6094 resulted in a dose- and time-dependent induction of cleaved PARP, a marker of cell death. Data presented provide the first report testing therapeutics in preclinical in vivo models of PRCC and support the clinical development of AZD6094 in this indication.

Abstract A217: Comparison of long-term pharmacodynamic actions of the synthetic small molecule HSP90 inhibitor AT13387 in multiple xenograft models

Abstract AT13387 is a novel small molecule inhibitor of HSP90. a member of a family of molecular chaperones. Previously we highlighted an association between the high affinity binding of AT13387 to the N-terminal ATPase domain of HSP90 and the duration of target inhibition in tumor cell lines in vitro. Further, AT13387 was shown to inhibit HSP90 and deplete client proteins in tumor xenografts longer than other, lower affinity inhibitors in the class. Here we have expanded the investigation to a wider number of tumor cell lines and to in vivo xenograft models and demonstrate that AT13387 has an extended pharmacodynamic action in tumors compared to other HSP90 inhibitors. We reason that the cumulative effects of these properties allow for less frequent dosing thus maximising efficacy whilst minimising systemic exposure and the potential for side effects. This study reports extended inhibition of HSP90 by AT13387 in a wider range of tumor cell lines in vitro. A 24hr exposure of A375 (melanoma) cells to AT13387 suppressed the expression of client proteins for 72 hrs or more. However in other cell lines such as NCI-H1975 (lung) and BT474 (breast), the suppression of client proteins by AT13387 was found to last in excess of 7 days. The pharmacodynamic action of AT13387 in vivo has been compared with that of 17-AAG and SNX-5422 in A375 and NCI-H1975 xenografts in nude mice. Following a single dose of each agent, we have investigated and compared the time course of the suppression of levels of several client proteins (e.g. AKT, CDK4) and the phosphorylation of key growth/survival signalling components (e.g. pERK, pS6, pAKT). These effects were rapidly induced in tumors following treatment with AT13387 and levels remained suppressed for up to 96 hrs. The durability of the AT13387 effects was significantly greater than for the other competitor compounds. Investigation of tumor growth in these models demonstrated that the longer pharmacodynamic action of AT13387 ensured that efficacy could be maintained on a once weekly schedule, whereas such a schedule for the other agents resulted in a significant loss of their anti-tumor effects. These data provide further support for the potential benefit of long acting HSP90 inhibitors as a way of maintaining anti-tumor effects whilst minimising potential for undesirable effects associated with systemic exposure. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A217.

Down‐regulation of the Notch pathway mediated by a γ‐secretase inhibitor induces anti‐tumour effects in mouse models of T‐cell leukaemia

gamma-Secretase inhibitors (GSIs) block NOTCH receptor cleavage and pathway activation and have been under clinical evaluation for the treatment of malignancies such as T-cell acute lymphoblastic leukaemia (T-ALL). The ability of GSIs to decrease T-ALL cell viability in vitro is a slow process requiring >8 days, however, such treatment durations are not well tolerated in vivo. Here we study GSI's effect on tumour and normal cellular processes to optimize dosing regimens for anti-tumour efficacy. Inhibition of the Notch pathway in mouse intestinal epithelium was used to evaluate the effect of GSIs and guide the design of dosing regimens for xenograft models. Serum Abeta(40) and Notch target gene modulation in tumours were used to evaluate the degree and duration of target inhibition. Pharmacokinetic and pharmacodynamic correlations with biochemical, immunohistochemical and profiling data were used to demonstrate GSI mechanism of action in xenograft tumours. Three days of >70% Notch pathway inhibition was sufficient to provide an anti-tumour effect and was well tolerated. GSI-induced conversion of mouse epithelial cells to a secretory lineage was time- and dose-dependent. Anti-tumour efficacy was associated with cell cycle arrest and apoptosis that was in part due to Notch-dependent regulation of mitochondrial homeostasis. Intermittent but potent inhibition of Notch signalling is sufficient for anti-tumour efficacy in these T-ALL models. These findings provide support for the use of GSI in Notch-dependent malignancies and that clinical benefits may be derived from transient but potent inhibition of Notch.