Optimization of isoquinolinone PI3K inhibitors led to the discovery of a potent inhibitor of PI3K-γ (26 or IPI-549) with >100-fold selectivity over other lipid and protein kinases. IPI-549 demonstrates favorable pharmacokinetic properties and robust inhibition of PI3K-γ mediated neutrophil migration in vivo and is currently in Phase 1 clinical evaluation in subjects with advanced solid tumors.

Background & Aims: Fibrolamellar carcinoma (FLC) is a rare, difficult-to-treat liver cancer primarily affecting pediatric and adolescent patients, and for which precision medicine approaches have historically not been possible. The DNAJB1-PRKACA gene fusion was identified as a driver of FLC pathogenesis. We aimed to assess whether FLC tumors maintain dependency on this gene fusion and determine if PRKACA is a viable therapeutic target. Methods: FLC patient-derived xenograft (PDX) shRNA cell lines were implanted subcutaneously into female NOD-SCID mice and tumors were allowed to develop prior to randomization to doxycycline (to induce knockdown) or control groups. Tumor development was assessed every 2 days. To assess the effect of treatment with novel selective PRKACA small molecule kinase inhibitors, BLU0588 and BLU2864, FLC PDX tumor cells were implanted subcutaneously into NOD-SCID mice and tumors allowed to develop. Mice were randomized to treatment (BLU0588 and BLU2864, orally, once daily) or control groups and tumor size determined as above. Results: Knockdown of DNAJB1-PRKACA reversed a FLC-specific gene signature and reduced PDX tumor growth in mice compared to the control group. Furthermore, FLC PDX tumor growth was significantly reduced with BLU0588 and BLU2864 treatment versus control (P = 0.003 and P = 0.0005, respectively). Conclusions: We demonstrated, using an inducible knockdown and small molecule approaches, that FLC PDX tumors were dependent upon DNAJB1-PRKACA fusion activity. In addition, this study serves as a proof-of-concept that PRKACA is a viable therapeutic target for FLC and warrants further investigation.

Hematopoietic progenitor kinase 1 (HPK1, MAP4K1) is a serine/threonine kinase that has been demonstrated to have suppressive effects across a range of immune cells, including T cells and dendritic cells. Loss of MAP4K1 kinase activity is sufficient to enhance T cell receptor (TCR) signaling resulting in robust anti-tumor immunity alone and in combination with checkpoint inhibition. These data support that MAP4K1 is a novel and attractive target for cancer immunotherapy. We have designed a series of potent, selective, and orally bioavailable inhibitors of MAP4K1. Treatment of primary human T cells or peripheral blood with either BLU2069 or BLU6348 was able to inhibit phosphorylation of pSLP76, a scaffolding protein that regulates MAPK downstream of the TCR. In addition, we show that compound treatment can enhance cytokine secretion and proliferation in human T cells in response to TCR crosslinking. The therapeutic benefit of MAP4K1 inhibition alone and in combination with anti-PD-L1 was evaluated in multiple syngeneic mouse tumor models including MCA205, MC38 and EMT-6. Treatment with either compound alone led to a reduction in tumor growth that was further enhanced when combined with anti-PD-L1 therapy. When tumors were grown in immunocompromised mice (MCA-205) or in the setting of CD8+ T cell depletion (MC-38), the anti-tumor effect of BLU2069 and BLU6348 respectively was lost, confirming the importance of immune cells in compound mediated antitumor effects. We further show that MCA205 tumors harvested from mice treated with BLU2069 had increased intratumoral CD8+ T cell infiltration, resulting in enhanced CD8/Treg ratios. In addition, transcriptional analysis of tumor lysates showed that BLU2069 significantly increased genes associated with an effector phenotype. These data support that pharmacological inhibition of MAP4K1 reduced tumor burden and enhanced antitumor immunity in preclinical tumor models. Finally, we show that MAP4K1 inhibition can enhance CD3/CD28-induced IL2 and IFNγ in human tumor infiltrating lymphocytes (TILs) generated from melanoma or non-small cell lung cancer (NSCLC) primary tumors. This work describes the identification of potent small molecule inhibitors of MAP4K1 which could be novel therapeutic agents and induce an effective immune response either alone or in combination with approved checkpoint inhibitors. Citation Format: Kerrie Faia, Alberto Toso, Kristina Fetalvero, Marly Roche, Steven Bench, Erin O'Hearn, Qiongfang Cao, Kerry-Ann Bright, Debora Paduraru, Andrea Romagnani, Weifan Weng, Tina Zimmermann, Michael Burke, Joshua Close, Luke Green, Joseph Kim, Chandra Miduturu, Alison Ribeiro, Marina Bacac, Sylvia Herter, Emanuele Perola, Michael Sheets, Jan Eckmann, Gordon Heidkamp, Tary Traore, Erik Gerson, Rich Woessner, Carsten Wolter, Felix Scheuplein, Nisha Perez, Timothy LaBranche, Grace Silva, Chaoyang Ye, Caitlin Utt, Stefan Gross, James R. Bischoff, Marion Dorsch, Tim Guzi, Klaus Hoeflich, Jason Brubaker. MAP4K1 inhibition enhances immune cell activation and anti-tumor immunity in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1717.

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Introduction: Fibrolamellar Carcinoma (FLC) is a rare primary liver malignancy, affecting children and young adults without chronic liver disease. FLC tumors are largely resistant to chemotherapy, making the identification of effective treatment options urgently needed. Recent genomic data strongly suggest that DNAJB1-PRKACA kinase fusions are the drivers of the vast majority of FLC cases. However, it has not been assessed whether FLC tumors remain dependent on DNAJB1-PRKACA expression and whether PRKACA inhibition could be a therapeutic approach for FLC. Here we summarize the preclinical evaluation of PRKACA as a potential therapeutic target for FLC. Methods: We established a xenograft model from a FLC- patient and then developed inducible PRKACA shRNA cell lines from this model. We also designed potent tool compounds that selectively inhibit the PRKACA protein to assess PRKACA as a potential therapeutic target for FLC. Results: We characterized a patient-derived xenograft (PDX) model of FLC (LI5132) and confirmed DNAJB1-PRKACA fusion expression and constitutive PRKACA pathway activation measured by phospho-VASP. The model also shows fibrolamellar type histology by H&E staining and expression of typical FLC markers like cytokeratin 7 and CD68 by IHC. Using inducible PRKACA-specific shRNA cell lines from this PDX model we demonstrated that the FLC transcriptional gene signature correlates strongly with expression of the DNAJB1-PRKACAfusion protein. Importantly, we demonstrated for three inducible PRKACA shRNA cell-line-derived xenograft models that the in vivo tumor growth remained dependent on DNAJB1-PRKACA fusion expression (TGI-72%-78%, day 22). PRKACA knockdown tumors displayed reduced Ki67 index (6.4 %) when compared to non-induced controls (37.1 %) further confirming that proliferation of the tumors depends on the fusion expression. To investigate the PRKACA catalytic dependency of the FLC model, we designed potent and selective PRKACA inhibitors based on starting points from our proprietary kinase inhibitor library. These investigational compounds are the first selective and potent PRKACA inhibitors and provide excellent tools to assess in vitro and in vivo PRKACA dependency. These compounds achieved potent PRKACA pathway inhibition and dose-dependent inhibition of FLC-specific gene expression, including genes such as carbamoyl phosphate synthetase (CPS1) and forkhead box C1 (FoxC1). We established a pharmacokinetic/ pharmacodynamic relationship and demonstrated in vivo PRKACA pathway inhibition in PDX tumors, as measured by phospho-VASP. Importantly, oral delivery of a potent and selective PRKACA inhibitor achieved up to 80% PRKACA kinase inhibition and led to statistically significant FLC tumor growth inhibition (54%, day 34) on a tolerated schedule. These data demonstrate that FLC depends on PRKACA kinase activity. Conclusion: This study is the first evaluation of PRKACA kinase inhibition as a therapeutic approach for FLC. The results from these preclinical experiments provide strong evidence that FLC depends on PRKACA catalytic activity and that novel PRKACA inhibitors may significantly decrease tumor growth in vivo. Citation Format: Stefanie S Schalm, Erin O’Hearn, Kevin Wilson, Timothy LaBranche, Grace Silva, Lucian DiPietro, Neil Bifulco, Richard Woessner, Nicolas Stransky, Darshan Sappal, Adam Shutes, Robert Campbell, Riadh Lobbardi, Michael Palmer, Joseph Kim, Stephen Miller, Marion Dorsch, Christoph Lengauer, Timothy Guzi, Vivek Kadambi, Andrew Garner, Klaus P Hoeflich. Evaluating PRKACA as a therapeutic target for Fibrolamellar Carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B127. doi:10.1158/1535-7163.TARG-19-B127