After L858R and ex19del epidermal growth factor receptor (EGFR) mutations, ex20ins mutations are the third most common class of driver-mutations in non-small cell lung cancer (NSCLC). Unfortunately, first-, second-, and third-generation EGFR tyrosine kinase inhibitors (TKIs) are generally ineffective for ex20ins patients due to insufficient mutant activity and selectivity over wild-type EGFR, leading to dose-limiting toxicities. While significant advances in recent years have been made toward identifying potent EGFR ex20ins mutant inhibitors, mutant vs wild-type EGFR selectivity remains a significant challenge. STX-721 (53) is a potent, irreversible inhibitor of the majority of EGFR/HER2 ex20ins mutants and demonstrates excellent mutant vs wild-type selectivity both in vitro and in vivo. STX-721 is currently in phase 1/2 clinical trials for EGFR/HER2 ex20ins-driven NSCLC.

TPS3173 Background: Mutations in exon 20 of the EGFR gene account for approximately 4% to 10% of all EGFR mutations in Non-Small Cell Lung Cancer (NSCLC). Most of these mutations are insertions (EGFR ex20ins) that reduce the binding of first, second, and third generation tyrosine kinase inhibitors (TKI) to the ATP-binding pocket of the EGFR. Amivantamab, a bispecific anti-EGFR/c-MET-receptor antibody, is approved for the treatment of NSCLC with EGFR ex20ins mutations. However, there is significant unmet need for new oral agents that lack the limitations of intravenous administration and associated infusion-related toxicities and possess improved target engagement, mutant selectivity, and tolerability. PFL-721/STX-721 is an orally bioavailable, irreversible small-molecule inhibitor targeting a broad range of EGFR- and HER2-activating ex20ins mutations. PFL-721/STX-721 is highly selective for EGFR ex20ins mutations compared to wild type EGFR and exhibits greater selectivity compared to other EGFR mutant inhibitors. In addition, PFL-721/STX-721 has demonstrated superior anti-proliferation and antitumor effects compared to other investigational anti-EGFR ex20ins agents in relevant tumor models in vitro and in vivo. These observations suggest a more robust clinical risk-to-benefit profile and support further clinical investigation of PFL-721/STX-721. Methods: PFL-721/STX-721-101 (NCT06043817) is an open-label, first-in-human (FIH), Phase 1/2 study evaluating the safety, tolerability, pharmacokinetic (PK) exposure, and preliminary antitumor activity of PFL-721/STX-721 in participants with locally advanced or metastatic NSCLC harboring EGFR/HER2 ex20ins mutations. It consists of 3 parts: Part 1 Dose Escalation, Part 2 Recommended Phase 2 Dose (RP2D) selection, and Part 3 Dose Expansion. In Part 1, participants with NSCLC harboring EGFR or HER2 ex20ins mutations will be enrolled into sequential cohorts to receive ascending oral doses of PFL-721/STX-721 administered daily in 28-day treatment cycles. The main goal is to identify the maximum tolerated dose (MTD) and optimal biological dose (OBD) of PFL-721/STX-721. In Part 2, participants with NSCLC harboring EGFR ex20ins mutations who have received 1 to 2 prior lines of treatment, including a platinum-containing chemotherapy regimen and excluding EGFR targeted therapies with the exception of amivantamab, will be randomized 1:1 to receive PFL-721/STX-721 at the MTD or OBD in order to determine the optimal RP2D. Finally, Part 3 will further test the anticancer efficacy of PFL-721/STX-721 is administered at the RP2D. PFL-721/STX-721-101 is actively enrolling at 18 sites in 7 countries globally. Clinical trial information: NCT06043817 .

Abstract Background: EGFR mutations are well validated clinical targets in non-small cell lung cancer (NSCLC). Osimertinib, a highly-selective EGFR mutation-targeting covalent drug, is increasingly used in the first line setting for patients with NSCLC bearing EGFR L858R mutation or exon 19 deletions (ex19del). In a subset of these patients, co-occurring L858R/C797x or ex19del/C797x mutations (“double mutants”) are emerging as an on-target resistance mechanism, necessitating the need for new therapies, particularly in patients with CNS metastases. Materials and methods: STX-241 was tested across a panel of in vitro biochemical, cell signaling, and proliferation assays for potency against EGFR L858R and ex19del single mutants and the corresponding C797S double mutants. Additionally, STX-241 was tested for in vivo activity in mice bearing human NSCLC cell line xenografts NCI-H3255 (L858R) and PC-9 (ex19del), and in a PC-9-derived ex19del/C797S double mutant knock-in xenograft. Free CNS penetration (Kp,uu) was determined in non-tumor bearing mice. Osimertinib and gefitinib (an approved reversible EGFR inhibitor) were used as benchmark molecules across assays. Results: STX-241, an ATP-competitive reversible EGFR inhibitor representing novel chemical matter, demonstrated potent and selective inhibition of recombinant EGFR L858R/C797S mutant protein relative to wild-type, with increased mutant residence time relative to gefitinib. Potent (high picomolar to low nanomolar) inhibition of L858R, ex19del, L858R/C797S, and ex19del/C797S mutants was observed in proliferation assays using engineered Ba/F3 and human NSCLC cell lines. STX-241 demonstrated >150x selectivity for all tested EGFR mutants relative to wild-type EGFR in engineered Ba/F3 cells and human cancer cells. In these assays, STX-241 selectivity exceeded that of the benchmark reversible EGFR inhibitor gefitinib. Strong potency and double mutant selectivity was also observed for STX-241 in pharmacodynamic assays measuring EGFR pathway activation (pEGFR). STX-241 was well tolerated at doses of 15 mg/kg BID or 50 mg/kg QD in mice, where regression of EGFR exon 19 or 21 mutant NSCLC xenografts was observed, concomitant with EGFR pathway suppression. Notably, using an isogenic pair of PC-9 (EGFR ex19del) NSCLC xenografts that differ only by the presence of a C797S mutation, STX-241 demonstrates no drop off in antitumor activity in the presence of C797S. In mouse pharmacokinetic studies measuring free CNS penetration, Kp,uu measurements were comparable to Osimertinib, run in parallel as a benchmark. Conclusions: STX-241 demonstrates strong potency and selectivity against EGFR L858R/C797S and ex19del/C797S double mutants, as well as robust CNS penetrance. These data warrant further exploration of this potential best-in-class inhibitor in the clinic for patients progressing on Osimertinib via C797x mutation. Citation Format: Raymond A Pagliarini, Benjamin C Milgram, Deanna R Borrelli, Erin O'Hearn, Michael R. Huff, Brendon Ladd, Natasja Brooijmans, Weixue Wang, Petr Kuzmic, Angel Guzman-Perez, Darrin D Stuart. Identification of STX-241, a CNS-penetrant and mutant-selective EGFR inhibitor with activity on osimertinib-resistant C797x mutations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B168.

PDF file - 1496K, Figure S1 - control expression and SMM hits representation; luciferase assay with MITF for BRD32048 and luciferase assay using BRD32048 analogs. Figure S2 - SPR data control graphs for TBX21 reference experiment. Figure S3 alternative SPR data for antibody reference; RU signals for additional reference surfaces. Figure S4 - ETV1 silencing validation data at mRNA and protein levels; alternative view of LNCaP signatures overlap; the proliferative effects of BRD32048 in LNCaP cell line and QPCR validation of BRD32048 inhibited targets. Figure S5 - control expression of indicated proteins and control invasion assays; - proliferation of cell lines in the presence of BRD32048. Figure S6 supportive mechanistic evidence such as ETV1-DNA interaction; BRD32048-induced loss of endogenous ETV1 and the effect of BRD32048 on an acetylation-deficient ETV1 mutant.

PDF file - 93KB, Estimates Fluxes for HCT116 Parental and IDH1 R132H/+ 2H1 cells under Normoxia and Hypoxia (2 percent Oxygen).

PDF file - 348KB, Figure S1: Isogenic IDH1 mutation compromises metabolic reprogramming under hypoxia. Figure S2: Simulated and measured uncorrected MIDs. Figure S3: Compromised Reductive TCA Metabolism is specific to cells with mutant IDH1. Figure S4: Cells with endogenous IDH1 and IDH2 mutations respond differently to mitochondrial stress. Figure S5: Inhibition of mutant IDH1 does not rescue reprogramming of TCA metabolism. Figure S6: Cells expressing mutant IDH1 are sensitive to pharmacological inhibition of mitochondrial oxidative metabolism.

Supplementary Table from Blocking PI3K p110β Attenuates Development of PTEN-Deficient Castration-Resistant Prostate Cancer

Supplementary Methods, Figures 1-5 from PIK3CA Mutation Uncouples Tumor Growth and Cyclin D1 Regulation from MEK/ERK and Mutant KRAS Signaling

Abstract A common outcome of androgen deprivation in prostate cancer therapy is disease relapse and progression to castration-resistant prostate cancer (CRPC) via multiple mechanisms. To gain insight into the recent clinical findings that highlighted genomic alterations leading to hyperactivation of PI3K, we examined the roles of the commonly expressed p110 catalytic isoforms of PI3K in a murine model of Pten-null invasive CRPC. While blocking p110α had negligible effects in the development of Pten-null invasive CRPC, either genetic or pharmacologic perturbation of p110β dramatically slowed CRPC initiation and progression. Once fully established, CRPC tumors became partially resistant to p110β inhibition, indicating the acquisition of new dependencies. Driven by our genomic analyses highlighting potential roles for the p110β/RAC/PAK1 and β-catenin pathways in CRPC, we found that combining p110β with RAC/PAK1 or tankyrase inhibitors significantly reduced the growth of murine and human CRPC organoids in vitro and in vivo. Because p110β activity is dispensable for most physiologic processes, our studies support novel therapeutic strategies both for preventing disease progression into CRPC and for treating CRPC. Implications: This work establishes p110β as a promising target for preventing the progression of primary PTEN-deficient prostate tumors to CRPC, and for treating established CRPC in combination with RAC/PAK1 or tankyrase inhibitors.

There is a compelling need for new therapeutic strategies for glioblastoma multiforme (GBM). Preclinical target and therapeutic discovery for GBMs is primarily conducted using cell lines grown in serum-containing media, such as U-87 MG, which do not reflect the gene expression profiles of tumors found in GBM patients. To address this lack of representative models, we sought to develop a panel of patient-derived GBM models and characterize their genomic features, using RNA sequencing (RNA-seq) and growth characteristics, both when grown as neurospheres in culture, and grown orthotopically as xenografts in mice. When we compared these with commonly used GBM cell lines in the Cancer Cell Line Encyclopedia (CCLE), we found these patient-derived models to have greater diversity in gene expression and to better correspond to GBMs directly sequenced from patient tumor samples. We also evaluated the potential of these models for targeted therapy, by using the genomic characterization to identify small molecules that inhibit the growth of distinct subsets of GBMs, paving the way for precision medicines for GBM.

Despite considerable efforts to identify cancer metabolic alterations that might unveil druggable vulnerabilities, systematic characterizations of metabolism as it relates to functional genomic features and associated dependencies remain uncommon. To further understand the metabolic diversity of cancer, we profiled 225 metabolites in 928 cell lines from more than 20 cancer types in the Cancer Cell Line Encyclopedia (CCLE) using liquid chromatography-mass spectrometry (LC-MS). This resource enables unbiased association analysis linking the cancer metabolome to genetic alterations, epigenetic features and gene dependencies. Additionally, by screening barcoded cell lines, we demonstrated that aberrant ASNS hypermethylation sensitizes subsets of gastric and hepatic cancers to asparaginase therapy. Finally, our analysis revealed distinct synthesis and secretion patterns of kynurenine, an immune-suppressive metabolite, in model cancer cell lines. Together, these findings and related methodology provide comprehensive resources that will help clarify the landscape of cancer metabolism.

Interferons (IFNs) are cytokines that play a critical role in limiting infectious and malignant diseases(1-4). Emerging data suggest that the strength and duration of IFN signaling can differentially impact cancer therapies, including immune checkpoint blockade(5-7). Here, we characterize the output of IFN signaling, specifically IFN-stimulated gene (ISG) signatures, in primary tumors from The Cancer Genome Atlas. While immune infiltration correlates with the ISG signature in some primary tumors, the existence of ISG signature-positive tumors without evident infiltration of IFN-producing immune cells suggests that cancer cells per se can be a source of IFN production. Consistent with this hypothesis, analysis of patient-derived tumor xenografts propagated in immune-deficient mice shows evidence of ISG-positive tumors that correlates with expression of human type I and III IFNs derived from the cancer cells. Mechanistic studies using cell line models from the Cancer Cell Line Encyclopedia that harbor ISG signatures demonstrate that this is a by-product of a STING-dependent pathway resulting in chronic tumor-derived IFN production. This imposes a transcriptional state on the tumor, poising it to respond to the aberrant accumulation of double-stranded RNA (dsRNA) due to increased sensor levels (MDA5, RIG-I and PKR). By interrogating our functional short-hairpin RNA screen dataset across 398 cancer cell lines, we show that this ISG transcriptional state creates a novel genetic vulnerability. ISG signature-positive cancer cells are sensitive to the loss of ADAR, a dsRNA-editing enzyme that is also an ISG. A genome-wide CRISPR genetic suppressor screen reveals that the entire type I IFN pathway and the dsRNA-activated kinase, PKR, are required for the lethality induced by ADAR depletion. Therefore, tumor-derived IFN resulting in chronic signaling creates a cellular state primed to respond to dsRNA accumulation, rendering ISG-positive tumors susceptible to ADAR loss.

Mutant isocitrate dehydrogenase 1 (IDH1) is an attractive therapeutic target for the treatment of various cancers such as AML, glioma, and glioblastoma. We have evaluated 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors that bind to an allosteric, induced pocket of IDH1R132H. This Letter describes SAR exploration focused on improving both the in vitro and in vivo metabolic stability of the compounds, leading to the identification of 19 as a potent and selective mutant IDH1 inhibitor that has demonstrated brain penetration and excellent oral bioavailability in rodents. In a preclinical patient-derived IDH1 mutant xenograft tumor model study, 19 efficiently inhibited the production of the biomarker 2-HG.

Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1R132H mutation destabilizes an IDH1 "regulatory segment," which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.

Inhibition of mutant IDH1 is being evaluated clinically as a promising treatment option for various cancers with hotspot mutation at Arg(132). Having identified an allosteric, induced pocket of IDH1(R132H), we have explored 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors for in vivo modulation of 2-HG production and potential brain penetration. We report here optimization efforts toward the identification of clinical candidate IDH305 (13), a potent and selective mutant IDH1 inhibitor that has demonstrated brain exposure in rodents. Preclinical characterization of this compound exhibited in vivo correlation of 2-HG reduction and efficacy in a patient-derived IDH1 mutant xenograft tumor model. IDH305 (13) has progressed into human clinical trials for the treatment of cancers with IDH1 mutation.

Introduction: Isocitrate dehydrogenase (IDH) enzymes catalyze the NADP-dependent interconversion of isocitrate and α-ketoglutarate. R132* IDH1 mutations lead to cellular accumulation of 2-hydroxyglutarate (2-HG), an oncometabolite that promotes tumorigenesis. IDH1 mutations are found in glioma (~80%), chondrosarcoma (~50%), cholangiocarcinoma (~20% intrahepatic), acute myeloid leukemia (AML; ~6-9%), and myelodysplastic syndrome (MDS; ~3%). IDH305 is a potent, orally available, mutant-selective, allosteric IDH1 inhibitor. IDH305 suppresses mutant IDH1-dependent 2-HG production and cell proliferation with an IC50 of 24 nM, and has antitumor activity in preclinical studies.