Notable large-scale immuno-oncology consortia formed in the recent era.

MANIFEST Consortium List of Authors

Circulating tumor cells (CTCs) and their clusters are the cellular drivers of metastasis. This study uses microfluidic models mimicking human capillary bifurcations to better understand how these cells interact with capillary beds. Patient CTCs, CTC-derived explant cells, and numerous cancer cell lines shed nuclei-free fragments in a cell size- and bifurcation-dependent manner. These shedding events, which reduces cell sizes up to 61%, facilitate CTC transit through bifurcations. The shed fragments are a novel subclass of large extracellular vesicles (LEVs) that we name "shearosomes" based on their requirement of shear stress for their biogenesis, and whose proteome is associated with immune-related pathways. Shearosomes exhibit functions that are characteristic of previously identified extracellular vesicles (EVs), including cell-directed internalization by endothelial and immune cells, and intercellular communication capabilities such as disruption of endothelial barrier integrity, polarization of monocytes toward M2 tumor-promoting macrophages, and mediating interactions between endothelial and immune cells. These findings suggest that CTCs shed shearosomes within capillary beds that affect cells implicated in the metastatic cascade.

Summary: Immunotherapy has revolutionized survival outcomes for many patients diagnosed with cancer. However, biomarkers that can reliably distinguish treatment responders from nonresponders, predict potential life-threatening and life-changing drug-induced toxicities, or rationalize treatment choices are still lacking. In response to this unmet clinical need, we introduce Multiomic ANalysis of Immunotherapy Features Evidencing Success and Toxicity, a tumor type–agnostic platform to provide deep profiling of patients receiving immunotherapy that will enable integrative identification of biomarkers and discovery of novel targets using artificial intelligence and machine learning.

Copper has been shown to play an important role in cancers, enhancing cell proliferation, remodelling the microenvironment and enhancing the function of oncogenes. Here we show that copper turns the tumour suppressor p53 protein into a pro-oncogenic protein. Mutations in the TP53 gene often lead to expression of various mutant p53 proteins. Mutant proteins often lose some or all wild type function and gain novel pro-tumourigenic functions, which can be partially attributed to protein unfolding. Here we show that copper accumulates in tumours, unfolds WTp53 and promotes chemoresistance. Unfolding of WT p53 results in interaction with mutant p53-specific interacting proteins TAp63 and Ago2 and promotes invasion. Interestingly, partially-functional p53 mutants that are frequently observed in lung cancers, are more affected by copper than WTp53. Our results suggest that small increases in copper impair WT p53 function and augment mutant p53 function, which could partially be restored by the chelator Trien.

Abstract Background: RAS-driven cancers represent a significant unmet clinical need, while breakthroughs in treatments targeting KRASG12C driven-NSCLC have invigorated drug-development for other RAS mutant cancers. Most RAS mutant cancers are not driven by KRASG12C, leaving an enormous amount of work needed to establish the contextual treatment vulnerabilities of cancers driven by different RAS mutant subtypes. RAS-Bio was pioneered within The Cancer Research UK Lung Cancer Centre of Excellence in Manchester. RAS-Bio is a uniquely curated resource of unparalleled clinical, genomic, translational and biological detail. RAS-Bio was established with the aim of generating novel model systems along with clinical observation to facilitate RAS precision medicine breakthroughs. Method: RAS-Bio recruits patients affected by epithelial cancers with RAS pathway mutations, aiming to i) optimize sample collection & processing protocols (FFPE/fresh/frozen) for multi-omic profiling, ii) validate preclinical hypothesis-generating findings in a clinical cohort, iii) generate novel RAS-mutant organoid, PDX and other models for analyses and drugging experiments, iv) foster academic/industry collaboration using a unique dataset including demographics, pathology/imaging details, treatment types and survival outcomes. Results: In two years, we have recruited 190 cancer patients with cancers harboring KRASG12C (n=71), KRASG12D(17), KRASG12V(23), KRASG12A (8), KRASG12S (2), KRASG12F (3), KRASG12R (1) and KRASG12E (1). A further 20 harboring KRAS codon 13 mutations and 12 KRASQ61 cancers and an additional 32 patients with mutations in other key signaling nodes of the MAPK pathway have been recruited. Seventeen PDX models have been attempted; lung cancer (14) and colorectal cancer (3). Four lung cancer cases are currently growing (KRASG12C treatment naïve, KRASG12C inhibitor resistance, KRASG12C inhibitor refractory, KRASG13C treatment naïve) and two colorectal cancer cases (KRASG12C and KRASG12V). We will present two deep-dive lung patient cases i) KRASG12C ‘OFF’ state inhibitor (sotorasib) resistance where multiple tissue samples, CTCs and a successful PDX were derived, and ii) sotorasib resistance where paired biopsies and PDXs were derived pre- and post-treatment. Both cases demonstrate the clinical utility of longitudinal sampling in understanding clinically relevant resistance mechanisms to targeted treatment. Conclusion: RAS-Bio represents a comprehensive biobank of clinical, pathological, and genomic detail in RAS-mutant lung cancers. Optimizing collaborative potential with academia/industry to facilitate prospective sampling of patients at different timepoints and integrating colorectal and pancreas cancers in our protocol. Citation Format: Mathew Carter, Katherine D. Brown, Helen Adderley, George Morrissey, Laura Woodhouse, Jamie Weaver, Kathryn Simpson, Jordan Roebuck, Jane Rogan, Joseph Mercer, Anshuman Chaturvedi, David Wedge, Claus Jorgensen, Angeliki Malliri, Caroline Dive, Colin R. Lindsay. RAS-Bio a unique pan-cancer biobank for RAS-driven tumors [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 2494.

BackgroundPredictive biomarkers for immune checkpoint blockade (ICB) in the second-line (2L) treatment of metastatic renal cell carcinoma (mRCC) are lacking.MethodsPatients with histologically confirmed RCC who started nivolumab after at least four months of tyrosine kinase inhibitors (TKIs) were recruited for this study. Serial tissue and blood samples were collected for immune biomarker evaluation. The primary endpoint was to determine the association of specific T cell subsets with clinical outcomes tested using Wilcoxon rank sum for clinical benefit rate (CBR) and log-rank test for progression-free survival (PFS).ResultsTwenty patients were included in this trial with a median age of 64 years and followed-up for a median of 12 months. The median PFS for patients who received TKI was 13.8 months, while for those subsequently treated with nivolumab following TKI therapy, the median PFS was 2.6 months. CBR of nivolumab was 20% with two partial responses. Functionally active PD1+ CD4+ T cells were enriched in non-responders (q=0.003) and associated with worse PFS on nivolumab (p=0.04). Responders showed a significant reduction in the effector CD4+T cell (TEF) fraction compared to non-responders at 3 months on nivolumab (0.40 vs 0.80, p=0.0005). CD127+CD4+ T cells were enriched in patients who developed immune-related adverse effects (q=0.003). Using in-house validated multiplex immunohistochemistry for six markers, we measured tumour-associated immune cell densities in tissue samples. Responders to nivolumab showed a significantly higher mean of immune cell densities in tissue samples compared to non-responders (346 vs 87 cells/mm2, p= 0.04).ConclusionIn this small study, analysis of tissue-based and peripheral blood immune cell subsets predicted clinical outcomes of nivolumab. Further studies are warranted with larger populations to validate these observations.

Small cell lung cancer (SCLC) is highly aggressive with poor prognosis. Despite a relative prevalence of circulating tumour DNA (ctDNA) in SCLC, liquid biopsies are not currently implemented, unlike non-SCLC where cell-free DNA (cfDNA) mutation profiling in the blood has utility for guiding targeted therapies and assessing minimal residual disease. cfDNA methylation profiling is highly sensitive for SCLC detection and holds promise for disease monitoring and molecular subtyping; cfDNA fragmentation profiling has also demonstrated clinical potential. Extrachromosomal DNA (ecDNA), that is often observed in SCLC, promotes tumour heterogeneity and chemotherapy resistance and can be detected in blood. We discuss how these cfDNA profiling modalities can be harnessed to expand the clinical applications of liquid biopsy in SCLC.

PDF file - 74 KB, GDC-0941 inhibits hypoxic signaling in drug sensitive U87 tumors.

Supplementary Figure 1. Combination treatment with olaparib/AZD1775 is effective in select SCLC CDX models. Mice bearing CDX3 (a), CDX4 (b), CDX8 (c), or CDX8p (d) tumors were treated with vehicles (black) AZD1775 (red), olaparib (green), or the combination (blue) for 21 days. Each line depicts an individual relative tumor volume from all mice. e) CDX4 tumors were treated with vehicles (black) AZD1775 (red), topotecan (green), or the combination (blue). Topotecan was administered on days 1-3 and AZD1775 was administered on days 1-21. Supplementary Figure 2. CDX8p is more chemoresistant than CDX8. a) The clinical timeline for the patient that gave rise to CDX8/8p. Mice bearing CDX8 (b) or CDX8p (c) tumors were treated with vehicles (black) or cisplatin/etoposide (red). Individual relative tumor volumes are depicted. Supplementary Figure 3. Olaparib/AZD1775 is less effective after treatment with cisplatin/etoposide. Mice bearing CDX3 tumors were treated with cisplatin/etoposide on days 1-3 and allowed to recover for 3 weeks, at which time they were randomised and treated with vehicles (black) AZD1775 (red), olaparib (green), or the combination (blue) for 21days. The treatment periods are marked by orange lines. Individual relative tumor volumes are depicted for each mouse. Cohort sizes > 7. Supplementary Figure 4. AZD1775 and olaparib monotherapy activity in representative SCLC CDX models. CDX3 (a, b), CDX4 (c, d), CDX8 (e, f), or CDX8p (g, h) ex vivo cultures were treated with increasing concentrations of either AZD1775 (a, c, e, g) or olaparib (b, d, f, h) for 1 week and relative cell viability was assessed. One representative experiment of at least three is shown. Supplementary Figure 5. Representative immunohistochemical images for phospho-histone H3 (a), cleaved caspase 3 (b), phospho-CHK1 (c), and ï§H2AX (d) from CDX3 tumours treated with vehicle, AZD1775, olaparib, or the combination. Supplementary Figure 6. Lack of a BRCAness signature in olaparib-sensitive models. a) Heatmap of average log2 RPKM values are depicted for CDX3, CDX4, CDX8, and CDX8p following supervised clustering of models based on olaparib sensitivity. b) The number of synonymous (black) and nonsynonymous (white) mutations as determined by whole exome sequencing are graphed for CDX3, CD8, and CDX8p. The lack of a matched germline sample precluded somatic mutation calling in CDX4. c) Filtered RNAseq reads for PALB2 were loaded into the Broad Integrative Genomics Viewer and the E178* was highlighted in green for each read. Supplementary Figure 7. Published mechanisms of olaparib resistance. a) Lysates from two representative CDX tumors were immunoblotted for indicated proteins. b) PARP1 RNA expression was assessed in tumor lysates from CDX3, CDX4, DX8, and CDX8p. Three representative tumors from each model were examined. c) Lysates from two representative CDX tumors were immunoblotted for indicated proteins. Supplementary Figure 8. Treatment with AZD1775, olaparib, or the combination does not alter p21 expression. Duplicate tumor lysates taken 24 hours after indicated treatments were immunoblotted for indicated proteins. Supplementary Figure 9. Published mechanisms of AZD1775 resistance. a) SETD2 and PKMYT1 RNA expression was assessed in tumor lysates from CDX3, CDX4, CDX8, and CDX8p. Three representative tumors from each model were examined, and mean values are plotted against the cohort average maximal in vivo response to AZD1775. b) RRM2 levels were measured by immunoblotting lysates from CDX tumors harvested 2 hours after a single dose of vehicle (white), AZD1775 (red), olaparib (green), or the combination (blue). Samples were quantified and normalised to vinculin in each sample. Cohort sizes > 4 tumors per condition. Supplementary Table 1. Mass Specrometer and UPLC system parameters Supplementary Table 2. Optimization parameters for mass spectrometry analysis

Small cell lung cancer (SCLC) is an aggressive malignancy with critical need for new therapies. While currently treated as a single disease, SCLC is heterogenous, comprised of several transcriptional subtypes. Each of these subtypes has distinct drivers and may warrant unique therapeutic targets. Two potential therapeutic targets for SCLC are the pro-survival proteins BCL-2 and BCL-XL. BCL-2 is overexpressed in ASCL1 (A) and POUF3 (P) subtypes of SCLC. We therefore sought to evaluate efficacy of the dual BCL-2/XL inhibitor AZD0466 in SCLC models and to determine whether transcriptional subtype would predict response. AZD0466 is a novel drug-dendrimer conjugate. The active moiety, AZD4320, is a potent dual inhibitor of BCL-2 and BCL-XL. AZD4320 is covalently conjugated to a 5th-generation PEGylated poly-lysine dendrimer through a hydrolytically labile linker to make AZD0466. AZD0466 has been optimized to deliver efficacy while mitigating potential Cmax-driven on-target toxicities of AZD4320. AZD4320 was active (IC50 ≤0.1 µM) in 9/27 SCLC cell lines. AZD4320 in vitro sensitivity was enriched in cell lines that represented A and P subtypes of SCLC compared to NEUROD1 and YAP1 subtypes. We next profiled AZD0466 in a panel of SCLC patient-derived models: 14 patient-derived xenografts and 10 circulating tumor cell-derived xenografts. AZD0466 monotherapy dosed weekly IV was active in 12/24 SCLC xenografts, driving regressions in 8 models. AZD0466 drove efficacy and cleaved caspase-3 induction in a dose-dependent manner. Similar to in vitro, AZD0466 in vivo efficacy was enriched in subtype-A, driving responses in 10/14 ASCL1 models (7 regression, 3 stable disease). AZD0466 response also correlated strongly with BCL-2 mRNA expression (P<0.0001). AZD0466 outperformed the selective BCL-2 inhibitor venetoclax in 6/10 models. Notably, AZD0466 was active in models resistant to platinum/etoposide chemotherapy, the standard-of-care for SCLC. Together, these data suggest BCL-2/XL inhibition has therapeutic potential in SCLC. AZD0466 is in clinical development. The first-in-human study treated 9 patients with advanced solid tumors (NCT04214093) at doses from 50-200mg, all of which were well-tolerated. The BOR was SD observed in 3 patients (100mg) with 1 patient receiving treatment for 5.5 months. AZD0466 is now under evaluation in patients with hematologic malignancies (NCT04865419 and NCT05205161). AZD0466 has been dosed in 33 patients up to 2400mg. No DLTs have been reported to date. Initial clinical activity has been observed through reduction of bone marrow blasts following AZD0466 treatment. AZD0466 exhibits linear PK, consistent across solid tumor and leukemia patients. The doses tested are in line with preclinical studies in SCLC. Citation Format: Courtney L. Andersen, Giulia Fabbri, David Jenkins, Zumla Cader, Shringi Sharma, Areya Tabatabai, Srividya Balachander, Jordan Roebuck, Melanie Galvin, Kathryn Simpson, Caroline Dive, Jordi Rodon Ahnert, Jamal Saeh. AZD0466, a dual BCL-2/XL targeting nanomedicine, is active in small cell lung cancer models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6150.

Vasculogenic mimicry (VM) describes the formation of pseudo blood vessels constructed of tumor cells that have acquired endothelial-like properties. VM channels endow the tumor with a tumor-derived vascular system that directly connects to host blood vessels, and their presence is generally associated with poor patient prognosis. Here we show that the transcription factor, Foxc2, promotes VM in diverse solid tumor types by driving ectopic expression of endothelial genes in tumor cells, a process that is stimulated by hypoxia. VMproficient tumors are resistant to anti-angiogenic therapy, and suppression of Foxc2 augments response. This work establishes co-option of an embryonic endothelial transcription factor by tumor cells as a key mechanism driving VM proclivity and motivates the search for VM-inhibitory agents that could form the basis of combination therapies with anti-angiogenics.

Brain metastases represent an important clinical problem for patients with small-cell lung cancer (SCLC). However, the mechanisms underlying SCLC growth in the brain remain poorly understood. Here, using intracranial injections in mice and assembloids between SCLC aggregates and human cortical organoids in culture, we found that SCLC cells recruit reactive astrocytes to the tumour microenvironment. This crosstalk between SCLC cells and astrocytes drives the induction of gene expression programmes that are similar to those found during early brain development in neurons and astrocytes. Mechanistically, the brain development factor Reelin, secreted by SCLC cells, recruits astrocytes to brain metastases. These astrocytes in turn promote SCLC growth by secreting neuronal pro-survival factors such as SERPINE1. Thus, SCLC brain metastases grow by co-opting mechanisms involved in reciprocal neuron–astrocyte interactions during brain development. Targeting such developmental programmes activated in this cancer ecosystem may help prevent and treat brain metastases.

PDF file - 46 KB, Chronic dosing with GDC-0941 leads to an increase in MAP-kinase pathway signaling in sensitive tumors.

Introduction: Vasculogenic mimicry (VM), the process of tumor cell transdifferentiation to endow endothelial-like characteristics supporting de novo vessel formation, is associated with poor prognosis in several tumor types, including SCLC. In genetically engineered mouse models (GEMMs) of SCLC, NOTCH, and MYC co-operate to drive a neuroendocrine (NE) to non-NE phenotypic switch, and co-operation between NE and non-NE cells is required for metastasis. Here, we define the phenotype of VM-competent cells and molecular mechanisms underpinning SCLC VM using circulating tumor cell-derived explant (CDX) models and GEMMs. Methods: We analyzed perfusion within VM vessels and their association with NE and non-NE phenotypes using multiplex immunohistochemistry in CDX, GEMMs, and patient biopsies. We evaluated their three-dimensional structure and defined collagen-integrin interactions. Results: We found that VM vessels are present in 23/25 CDX models, 2 GEMMs, and in 20 patient biopsies of SCLC. Perfused VM vessels support tumor growth and only NOTCH-active non-NE cells are VM-competent in vivo and ex vivo, expressing pseudohypoxia, blood vessel develop-ment, and extracellular matrix organization signatures. On Matrigel, VM-primed non-NE cells remodel extracellular matrix into hollow tubules in an integrin b1-dependent process. Conclusions: We identified VM as an exemplar of functional heterogeneity and plasticity in SCLC and these findings take considerable steps toward understanding the molecular events that enable VM. These results support therapeutic co-targeting of both NE and non-NE cells to curtail SCLC progression and to improve the outcomes of patients with SCLC in the future. (c) 2023 International Association for the Study of Lung Cancer. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).

KRAS is the most frequently mutated oncogene in NSCLC, affecting ∼30% of adenocarcinomas. Resistance to novel KRAS G12C inhibitor monotherapies is inevitable and early data with inactive-state selective inhibitors suggest there are a diverse range of resistance mechanisms. A panel of G12C mutant NSCLC cell lines (H358, H23, HCC 1171, H1792, H2122) were cultured with an inactive state selective G12C inhibitor for over 9 months and resistant subclones generated. A multi-omic approach combining genomic, bulk RNA-seq, scRNA-seq and proteomic analyses was used to characterise these resistant populations, with the aim of identifying common resistance mechanisms and potential therapeutic vulnerabilities. In parallel, a PDX has been generated at the point of resistance to a G12C inhibitor, seeking to validate mechanistic insights. In resistant cell lines, there were varied morphological changes demonstrating both epithelial-mesenchymal-transition (EMT) and mesenchymal-epithelial-transition (MET). Bulk RNA-seq of two cell lines did not reveal common mechanisms of resistance. Further proteomic analysis suggests that there are a diverse range of resistance mechanisms present amongst the resistant populations. To better understand the transition to resistance, scRNA-seq of H1792 cells at different time points was performed, with different transcriptomic states identified. Work is ongoing to explore whether these states confer different sensitivities to G12C inhibition, and whether trajectory inference can predict a path to resistance. Analysis of a PDX has shown that it is a poorly differentiated adenocarcinoma of solid subtype, with further work exploring if this represents a histological transformation from pre-treatment samples. Therapeutic vulnerabilities to the resistant cell line panel were identified, with 5 cell lines remaining sensitive to active state selective RAS inhibitors. Resistance to inactive state selective KRAS G12C inhibitors is heterogeneous but resistant populations remain sensitive to RAS targeted therapies. These results suggest that KRAS mutant cell lines remain addicted to oncogenic KRAS in the setting of resistance.

PDF file - 75 KB, Uptake of 18F-FDG decreases significantly after ~18h of GDC-0941 treatment in U87, but not HCT116, tumors.

The Notch pathway is a conserved cell-cell communication pathway that controls cell fate decisions. Here we sought to determine how Notch pathway activation inhibits the neuroendocrine cell fate in the lungs, an archetypal process for cell fate decisions orchestrated by Notch signaling that has remained poorly understood at the molecular level. Using intratumoral heterogeneity in small-cell lung cancer as a tractable model system, we uncovered a role for the transcriptional regulators REST and YAP as promoters of the neuroendocrine to non-neuroendocrine transition. We further identified the specific neuroendocrine gene programs repressed by REST downstream of Notch in this process. Importantly, we validated the importance of REST and YAP in neuroendocrine to non-neuroendocrine cell fate switches in both developmental and tissue repair processes in the lungs. Altogether, these experiments identify conserved roles for REST and YAP in Notch-driven inhibition of the neuroendocrine cell fate in embryonic lungs, adult lungs, and lung cancer.