Richard Lee

Molecular modifiers of KRASG12C inhibitor (KRASG12Ci) efficacy in advanced KRASG12C-mutant NSCLC are poorly defined. In a large unbiased clinicogenomic analysis of 424 patients with non-small cell lung cancer (NSCLC), we identified and validated coalterations in KEAP1, SMARCA4, and CDKN2A as major independent determinants of inferior clinical outcomes with KRASG12Ci monotherapy. Collectively, comutations in these three tumor suppressor genes segregated patients into distinct prognostic subgroups and captured ∼50% of those with early disease progression (progression-free survival ≤3 months) with KRASG12Ci. Pathway-level integration of less prevalent coalterations in functionally related genes nominated PI3K/AKT/MTOR pathway and additional baseline RAS gene alterations, including amplifications, as candidate drivers of inferior outcomes with KRASG12Ci, and revealed a possible association between defective DNA damage response/repair and improved KRASG12Ci efficacy. Our findings propose a framework for patient stratification and clinical outcome prediction in KRASG12C-mutant NSCLC that can inform rational selection and appropriate tailoring of emerging combination therapies. SIGNIFICANCE: In this work, we identify co-occurring genomic alterations in KEAP1, SMARCA4, and CDKN2A as independent determinants of poor clinical outcomes with KRASG12Ci monotherapy in advanced NSCLC, and we propose a framework for patient stratification and treatment personalization based on the comutational status of individual tumors. See related commentary by Heng et al., p. 1513. This article is highlighted in the In This Issue feature, p. 1501.

For patients with advanced non-small-cell lung cancer (NSCLC), dual immune checkpoint blockade (ICB) with CTLA4 inhibitors and PD-1 or PD-L1 inhibitors (hereafter, PD-(L)1 inhibitors) is associated with higher rates of anti-tumour activity and immune-related toxicities, when compared with treatment with PD-(L)1 inhibitors alone. However, there are currently no validated biomarkers to identify which patients will benefit from dual ICB1,2. Here we show that patients with NSCLC who have mutations in the STK11 and/or KEAP1 tumour suppressor genes derived clinical benefit from dual ICB with the PD-L1 inhibitor durvalumab and the CTLA4 inhibitor tremelimumab, but not from durvalumab alone, when added to chemotherapy in the randomized phase III POSEIDON trial3. Unbiased genetic screens identified loss of both of these tumour suppressor genes as independent drivers of resistance to PD-(L)1 inhibition, and showed that loss of Keap1 was the strongest genomic predictor of dual ICB efficacy—a finding that was confirmed in several mouse models of Kras-driven NSCLC. In both mouse models and patients, KEAP1 and STK11 alterations were associated with an adverse tumour microenvironment, which was characterized by a preponderance of suppressive myeloid cells and the depletion of CD8+ cytotoxic T cells, but relative sparing of CD4+ effector subsets. Dual ICB potently engaged CD4+ effector cells and reprogrammed the tumour myeloid cell compartment towards inducible nitric oxide synthase (iNOS)-expressing tumoricidal phenotypes that—together with CD4+ and CD8+ T cells—contributed to anti-tumour efficacy. These data support the use of chemo-immunotherapy with dual ICB to mitigate resistance to PD-(L)1 inhibition in patients with NSCLC who have STK11 and/or KEAP1 alterations. Alterations in the tumour suppressor genes STK11 and/or KEAP1 can identify patients with advanced non-small-cell lung cancer who are likely to benefit from combinations of PD-(L)1 and CTLA4 immune checkpoint inhibitors added to chemotherapy.

Resistance to inactive state-selective RASG12C inhibitors frequently entails accumulation of RASGTP, rendering effective inhibition of active RAS potentially desirable. Here, we evaluated the antitumor activity of the RAS(ON) multiselective tricomplex inhibitor RMC-7977 and dissected mechanisms of response and tolerance in KRASG12C-mutant non-small cell lung cancer (NSCLC). Broad-spectrum reversible RASGTP inhibition with or without concurrent covalent targeting of active RASG12C yielded superior and differentiated antitumor activity across diverse comutational KRASG12C-mutant NSCLC mouse models of primary or acquired RASG12C(ON) or RASG12C(OFF) inhibitor resistance. Interrogation of time-resolved single-cell transcriptional responses established an in vivo atlas of multimodal acute and chronic RAS pathway inhibition in the NSCLC ecosystem and uncovered a regenerative mucinous transcriptional program that supports long-term tumor cell persistence. In patients with advanced KRASG12C-mutant NSCLC, the presence of mucinous histologic features portended poor response to sotorasib or adagrasib. Our results have potential implications for personalized medicine and the development of rational RAS inhibitor-anchored therapeutic strategies. Significance: Our work reveals robust and durable antitumor activity of the preclinical RAS(ON) multiselective inhibitor RMC-7977 against difficult-to-treat subsets of KRASG12C-mutant NSCLC with primary or acquired RASG12C inhibitor resistance and identifies a conserved mucinous transcriptional state that supports RAS inhibitor tolerance. See related commentary by Marasco and Misale, p. 2018.

Abstract Mistletoe extracts including Viscum Fraxini (extract of mistletoe growing on ash trees) have been used extensively in European medical clinics to treat various cancers including hepatocellular carcinoma (HCC). Results from a phase II clinical trial have shown that subcutaneous administration of Fraxini leads to either complete or partial response in 20% of chemo-naïve patients with HCC. However, how mistletoe extract exerts its antitumor activity in HCC is largely unknown. We examined the effect of Fraxini on the growth of human HCC Hep3B and HepG2 cells by MTT assay, apoptosis by PI and Annexin V staining and molecular mechanisms using Reverse Phase Proteomic Array (RPPA). All three mistletoe extracts, Fraxini, Iscador Q and M (growing on oak and maple trees, respectively), exerted relatively strong antiproliferative activity in the Hep3B cells, with IC50 values at 0.5, 6.49, and 5.7 µg/ml, respectively. A slightly weaker anticancer effect was observed in the HepG2 cells treated with the above three extracts. The antiproliferative effect of Fraxini was almost 10 and 6 times stronger than that of Iscador M and Iscador Q, respectively, in both Hep3B and HepG2 cells. Intriguingly, the water-soluble fraction of Fraxini exerted similar anticancer activity as Fraxini, but no inhibitory effect of lipid soluble fraction of Fraxini was observed. Treatment with Fraxini induced Hep3B cells to undergo apoptotic cell death, evidenced by the increased number (5-fold) of subG1/G0 phase cells in the Fraxini-treated (5 µg/ml) Hep3B cells than in the control cells and increased apoptotic cell population (by Annexin V staining). Additionally, notable ultrastructural changes i.e. extremely condensed mitochondria in perinuclear positions by transmission electron microscopy (TEM), were observed in Fraxini-treated (10 μg/ml) Hep3B cells compared to control treated cells. Proteomic array analysis suggested that Fraxini dose-dependently inhibited expression of Bcl-xl, BCl-2, Bim, and pRB proteins while increased cleaved caspase 7 and 8 as well as CHEK 1 and 2, suggesting that the apoptotic effect of Fraxini was mediated through regulation of BCL-2 family proteins and caspase activation, which correlated with the mitochondrial changes observed in the TEM study. Strikingly, c-Myc protein was strongly downregulated in a dose dependent fashion in the Hep3B cells treated with Fraxini (> 84% at 5 μg/ml), as examined by both RPPA and Western blotting, suggesting that the c-Myc protein could be an important target for Fraxini-elicited anticancer activity in HCC. Given that HCC is one of the few cancers whose death rate nearly equals its incidence and c-Myc is required for hepatocyte proliferation and liver tumorigenesis, Fraxini could have great potential in HCC treatment and therefore warrants further investigation. Citation Format: Xiaoping Ding, Carrie Cartwright, Lin Tan, Richard Lee, Peiying Yang. Mistletoe extract inhibits the proliferation of human hepatocellular carcinoma cells by induction of apoptosis and downregulation of c-MYC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3206. doi:10.1158/1538-7445.AM2014-3206