Introduction: Derazantinib (DZB), an inhibitor of the fibroblast growth factor receptors 1-3 (FGFR1-3), is in phase-2 clinical trials for cholangiocarcinoma, urothelial and gastric cancer for tumors with FGFR-amplifications, fusions and mutations. DZB has similar potency against colony-stimulating factor receptor-1 (CSF1R); a protein important in the function of M2 tumor-associated macrophages (M2-TAMs). M2-TAMs cause an immunosuppressive environment in solid tumors that decrease the effectiveness of checkpoint inhibitors such as PD-1/PD-L1 inhibitors, and promote angiogenesis and metastasis. Methods: Proliferating mouse macrophage cells (RAW264.7) and human tumor cells (GDM1 and DEL) were pre-treated for 1h with compounds. Cells were treated with/out CSF1 (10ng/ml) for 5min, lysed and analyzed by western blot. Images were quantified using the EvolutionCapt program. Murine 4T1 breast tumor cells were grown orthotopically in syngeneic Balb/c mice, Karpass299 lymphoma cells s.c. in CB17/Scid mice, and gastric (GA0119) and cholangiocarcinoma (CC6604)-PDX in athymic Balb/c mice. One 4T1 experiment used stably transfected luciferased (4T1-Luc) cells. DZB (65-75 mg/kg) and another FGFR-inhibitor, pemigatinib (1 mg/kg) were dosed daily, orally, and the murine PDL1-Antibody (PDL1-Ab) was dosed at 5-10 mg/kg, i.p., 2qw. Efficacy was assessed as a dT/C, and metastases were determined by counting nodules and/or bioluminescence ex vivo. Results: DZB inhibited p-CSF1R in cultured cells with mean±SEM IC50s of 307, <500, 54nM for RAW264.7, GDM1, DEL cells, respectively. These IC50s were similar to the potency of DZB against the HeLa cell-line transiently infected with FGFR2 (107nM), thus confirming kinase assays that indicated similar sensitivity of FGFR1-3 and CSF1R to DZB. Other FGFR-inhibitors, including pemigatinib, which has no CSF1R activity, were without significant effect. In 3 tumor xenograft models with reported expression/amplification of CSF1R, DZB had a mild inhibitory effect (ΔT/C=0.67±0.05), while pemigatinib had no efficacy (ΔT/C=1.02±0.16); mean±SEM. However, in these xenografts we were unable to detect p-CSF1R. In the FGFR-driven syngeneic tumor-model, 4T1, DZB was highly efficacious causing tumor-stasis. The PDL1-Ab was without efficacy in this model, but in combination with DZB, increased efficacy against the primary tumor, and more strongly against the liver and lung metastases. PD-analyses are ongoing to explore the mechanism of this interaction. Conclusion: DZB is a potent inhibitor of CSF1R in vitro. In combination with a murine PDL1-Ab, DZB showed a positive interaction in the orthotopic syngeneic breast tumor model, 4T1, against both the primary tumor and metastases. These data support clinical trials in which DZB is combined with the PDL1-Ab, atezolizumab (NCT04604132, NCT04045613). Citation Format: Mahmoud EL Shemerly, Laurenz Kellenberger, Elisa Zanini, Heidi Lane, Paul McSheehy. Derazantinib, an FGFR1-3 inhibitor, inhibits CSF1R in macrophages and tumor cell lines, and synergizes with a PDL1-antibody in an FGFR-driven murine syngeneic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5501.

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Background: BAL0891 is a dual inhibitor of threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1). These kinases collaborate in activating the mitotic spindle assembly checkpoint (SAC) at the kinetochore (KT) to regulate chromosome alignment and segregation prior to mitotic exit. In vitro, BAL0891 has a combined prolonged effect on TTK and a transient effect on PLK1, leading to rapid disruption of the SAC that potentiates aberrant mitotic progression of tumor cells. In this work, efficacy of BAL0891 was investigated in mouse models of human triple negative breast cancer (TNBC) including evaluation of dose-dependency, drug exposure, target occupancy and a screen of activity across a panel of PDX models. Methods: The MDA-MB-231 cell line was grown sc in nude mice and treated with BAL0891, administered IV weekly (QW) or twice-weekly (2QW). Thirteen sc TNBC PDX models were screened for BAL0891 response using 2QW administration. Efficacy was quantified as deltaT/C (treated/control tumors). Plasma and tumor were analyzed for drug levels or TTK target occupancy by LC-MS/MS. The latter used a biotinylated TTK-specific probe and streptavidin-mediated isolation of unoccupied TTK, trypsin digestion and quantification of TTK-representative peptides. Results: BAL0891 efficacy was tested in the TNBC xenograft model MDA-MB-231 with QW or 2QW IV dosing schedules. All treatments were well tolerated, with no drug-related animal deaths. With MTD dosing, tumor regressions were observed, while different MTD fractions for both QW and 2QW schedules showed dose-dependent anti-tumor activity. The weekly MTD group was followed for an additional 20 days after treatment cessation on day 100. Strikingly, 3 of 8 tumors continued to shrink resulting in 2 (25%) pathologically confirmed cures. Consistent with the potent efficacy of intermittent MTD dosing, and prolonged tumor drug exposure, tumor TTK was fully drug-occupied for ≥ 6 days after the last administration; target occupancy was also dose-and drug exposure-dependent. To further evaluate BAL0891 anti-cancer activity in TNBC, a screen in 13 TNBC PDX models was conducted. Seven models exhibited deltaT/C < 50%, with regressions observed in 3. Of these, 2 models showed persistent regressions ≥ 70% vs. baseline. Interestingly, evaluation of TTK target occupancy in selected models showed high target occupancy independent of tumor response, indicating target dependency rather than drug availability is important for anti-cancer activity. Conclusion: BAL0891 is a novel dual TTK/PLK1 mitotic checkpoint inhibitor with potent anti-cancer activity in TNBC models. Intermittent IV administration is well tolerated and associated with prolonged tumor drug exposure, prolonged TTK inhibition and notable anti-tumor efficacy. These data support further investigation of BAL0891 for the treatment of cancer patients (incl. TNBC). Citation Format: Heidi A. Lane, Felix Bachmann, Elisa Zanini, Paul McSheehy, Karine Litherland, Nicole Forster-Gross, Luc Bury, Diep Vu-Pham, Jos de Man, Wilhelmina E. van Riel, Guido JR Zaman, Rogier C. Buijsman, Laurenz Kellenberger. BAL0891: A novel dual TTK/PLK1 mitotic checkpoint inhibitor (MCI) that drives aberrant tumor cell division resulting in potent anti-cancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5645.

Derazantinib (DZB) is an inhibitor of the fibroblast growth factor receptors 1–3 (FGFRi) with similar potency against colony-stimulating factor receptor-1 (CSF1R), a protein important in the recruitment and function of tumor-associated macrophages. DZB inhibited pCSF1R in the macrophage cell line RAW264.7, and tumor cells GDM-1 and DEL, and had the same potency in HeLa cells transiently over-expressing FGFR2. DZB exhibited similar potency against pCSF1R expressed by isolated murine macrophages, but as in the cell lines, specific FGFRi were without significant CSF1R activity. DZB inhibited growth of three tumor xenograft models with reported expression or amplification of CSF1R, whereas the specific FGFRi, pemigatinib, had no efficacy. In the FGFR-driven syngeneic breast tumor-model, 4T1, DZB was highly efficacious causing tumor stasis. A murine PD-L1 antibody was without efficacy in this model, but combined with DZB, increased efficacy against the primary tumor and further reduced liver, spine and lung metastases. Immunohistochemistry of primary 4T1 tumors showed that the combination favored an antitumor immune infiltrate by strongly increasing cytotoxic T, natural killer and T-helper cells. Similar modulation of the tumor microenvironment was observed in an FGFR-insensitive syngeneic bladder model, MBT-2. These data confirm CSF1R as an important oncology target for DZB and provide mechanistic insight for the ongoing clinical trials, in which DZB is combined with the PD-L1 antibody, atezolizumab.

Background: BAL0891 is a dual inhibitor of threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1). These kinases collaborate in activating the mitotic spindle assembly checkpoint (SAC) at the kinetochore (KT) to regulate chromosome alignment and segregation prior to mitotic exit. In this work, kinase inhibition by BAL0891 was linked to effects on SAC integrity and aberrant mitotic progression in tumor cells. Comparison with a TTK-specific inhibitor (CFI-402257, CFI) allowed further evaluation of the contribution of dual TTK/PLK1 inhibition to anti-cancer activity, associated with a promising anti-proliferative profile across diverse tumor cell lines. Methods: Kinase assays used a radiometric assay. Target residency was measured using surface plasmon resonance with recombinant kinase. Anti-proliferative activity was assessed with crystal violet or YO-PRO assay (5 days incubation), in-cell target inhibition by immunoblotting for phospho-TTK following drug wash-out. Effects on SAC integrity were followed by immunoprecipitation (IP) mitotic progression by flow cytometry/mitotic marker expression. Cells were blocked in mitosis using the microtubule-targeting agent nocodazole or the PLK1 inhibitor onvansertib. SAC KT accumulation was evaluated by immunofluorescence (IF) for co-localization of BubR1 with CENPC. Comparative studies with CFI used anti-proliferative IC50 concentrations. Results: In vitro kinase profiling showed that BAL0891 has low nM IC50s against TTK and PLK1, with prolonged TTK (>12 h) and transient PLK1 (4 min) target residency. Prolonged TTK inhibition (≥38 h) was also observed in HT29 tumor cells. Consistent with a dominant TTK-targeting activity, BAL0891 treatment of HT29 cells blocked in mitosis with nocodazole or the PLK1 inhibitor onvansertib led to aberrant mitotic release and accumulation of polyploid cells. This was preceded by SAC disruption as visualized by IP assays. Effects on the SAC and mitotic exit were evaluated in comparative studies with CFI; BAL0891 exhibited faster kinetics for both parameters suggesting a contribution of PLK1 inhibition. This was confirmed by directly evaluating acute effects on SAC integrity at the KT by IF. Specifically, 1 h BAL0891 treatment of mitotic HT29 cells resulted in a highly reproducible and significant reduction in KT-associated SAC (p<0.0001) which was not observed with CFI in the same conditions. An extensive in vitro BAL0891 anti-proliferative screen indicated a broad anti-cancer potential, with low nM GI50s observed for most tumor lines and minimal activity on non-immortalized cells (GI50s >5 uM). Conclusion: BAL0891 is a novel dual TTK/PLK1 mitotic checkpoint inhibitor. In tumor cells, prolonged effects on TTK and transient effects on PLK1 contribute to rapid SAC disruption and aberrant mitotic exit. This is associated with potent anti-proliferative activity in diverse tumor lines. Citation Format: Elisa Zanini, Nicole Forster-Gross, Felix Bachmann, Nicole Willemsen-Seegers, Jos de Man, Guido J. Zaman, Rogier C. Buijsman, Anna Groner, Mila Roceri, Karin Burger, Paul McSheehy, Laurenz Kellenberger, Heidi A. Lane. BAL0891: A novel, small molecule, dual TTK/PLK1 mitotic checkpoint inhibitor (MCI) that drives aberrant tumor cell division [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5646.