Functional Heterogeneity in MET Pathway Activation in PDX Models of Osimertinib-resistant EGFR-driven Lung Cancer.

MET pathway activation is one of the most common mechanisms of resistance to osimertinib in EGFR-mutant non-small cell lung cancer (NSCLC). We previously demonstrated spatial and temporal heterogeneity in MET pathway activation upon osimertinib resistance in EGFR-mutant NSCLC; however, the functional relevance of these findings is unclear. Here, we generated 19 patient-derived xenografts (PDX) from 9 patients with multi-region and temporal sampling of osimertinib-resistant tumor tissue from patients with EGFR-mutant NSCLC. MET pathway activation was a putative mechanism of osimertinib resistance in 66% (n = 6/9) patients from whom PDXs were generated. Significant spatial and temporal heterogeneity in MET pathway activation was evident. Osimertinib-resistant PDXs with MET amplification by FISH (defined as MET/CEP7 ratio ≥2.0 or mean MET ≥ 6.0 copies/cell) and high-level phospho-MET, but not c-MET expression, had better responses to osimertinib and savolitinib combination than to osimertinib alone. MET polysomy tumors by FISH from both PDXs and patients had evidence of subclonal phospho-MET expression. Select MET polysomy PDX tumors with phospho-MET expression responded better to osimertinib and savolitinib combination than MET polysomy PDX tumors without phospho-MET expression. Our results suggest osimertinib and savolitinib combination is most effective for osimertinib-resistant EGFR-mutant tumors with MET pathway activation as evidenced by phospho-MET. As subclonal MET amplification may be evident in MET polysomy tumor progression, MET polysomy warrants close clinical follow-up with phospho-MET IHC in parallel with FISH diagnostic. SIGNIFICANCE: Using a novel cohort of in vivo PDX models of MET pathway activation with acquired resistance to osimertinib in EGFR-mutant lung cancer, we demonstrate that phospho-MET may be a clinically relevant assay to guide treatment selection with osimertinib and savolitinib combination. In addition, our work shows that patients with MET polysomy tumors may have subclonal MET amplification and therefore require close follow up for the use of osimertinib and savolitinib combination.

Translational Orthotopic Models of Glioblastoma Multiforme.

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumors displaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

Loss of Brca1 and Trp53 in adult mouse mammary ductal epithelium results in development of hormone receptor-positive or hormone receptor-negative tumors, depending on inactivation of Rb family proteins.

BACKGROUND: Breast cancer is a heterogenous disease with several histological and molecular subtypes. Models that represent these subtypes are essential for translational research aimed at improving clinical strategy for targeted therapeutics. METHODS: Different combinations of genetic aberrations (Brca1 and Trp53 loss, and inhibition of proteins of the Rb family) were induced in the mammary gland by injection of adenovirus expressing Cre recombinase into the mammary ducts of adult genetically engineered mice. Mammary tumors with different genetic aberrations were classified into molecular subtypes based on expression of molecular markers and RNAseq analysis. In vitro potency assays and Western blots were used to examine their drug sensitivities. RESULTS: Induction of Brca1 and Trp53 loss in mammary ductal epithelium resulted in development of basal-like hormone receptor (HR)-negative mammary tumors. Inhibition of Rb and Trp53 loss or the combination of Rb, Trp53 and Brca1 aberrations resulted in development of luminal ductal carcinoma positive for ER, PR, and Her2 expression. HR positivity in tumors with Rb, Trp53 and Brca1 aberrations indicated that functionality of the Rb pathway rather than Brca1 status affected HR status in these models. Mammary tumor gene expression profiles recapitulated human basal-like or luminal B breast cancer signatures, but HR-positive luminal cancer models were endocrine resistant and exhibited upregulation of PI3K signaling and sensitivity to this pathway inhibition. Furthermore, both tumor subtypes were resistant to CDK4/6 inhibition. CONCLUSIONS: Examination of molecular expression profiles and drug sensitivities of tumors indicate that these breast cancer models can be utilized as a translational platform for evaluation of targeted combinations to improve chemotherapeutic response in patients that no longer respond to hormone therapy or that are resistant to CDK4/6 inhibition.

Heterogeneity of neuroendocrine transcriptional states in metastatic small cell lung cancers and patient-derived models.

Molecular subtypes of small cell lung cancer (SCLC) defined by the expression of key transcription regulators have recently been proposed in cell lines and limited number of primary tumors. The clinical and biological implications of neuroendocrine (NE) subtypes in metastatic SCLC, and the extent to which they vary within and between patient tumors and in patient-derived models is not known. We integrate histology, transcriptome, exome, and treatment outcomes of SCLC from a range of metastatic sites, revealing complex intra- and intertumoral heterogeneity of NE differentiation. Transcriptomic analysis confirms previously described subtypes based on ASCL1, NEUROD1, POU2F3, YAP1, and ATOH1 expression, and reveal a clinical subtype with hybrid NE and non-NE phenotypes, marked by chemotherapy-resistance and exceedingly poor outcomes. NE tumors are more likely to have RB1, NOTCH, and chromatin modifier gene mutations, upregulation of DNA damage response genes, and are more likely to respond to replication stress targeted therapies. In contrast, patients preferentially benefited from immunotherapy if their tumors were non-NE. Transcriptional phenotypes strongly skew towards the NE state in patient-derived model systems, an observation that was confirmed in paired patient-matched tumors and xenografts. We provide a framework that unifies transcriptomic and genomic dimensions of metastatic SCLC. The marked differences in transcriptional diversity between patient tumors and model systems are likely to have implications in development of novel therapeutic agents.

Distinct Biomarker Profiles and TCR Sequence Diversity Characterize the Response to PD-L1 Blockade in a Mouse Melanoma Model.

Only a subset of patients responds to immune checkpoint blockade (ICB) in melanoma. A preclinical model recapitulating the clinical activity of ICB would provide a valuable platform for mechanistic studies. We used melanoma tumors arising from an Hgftg;Cdk4R24C/R24C genetically engineered mouse (GEM) model to evaluate the efficacy of an anti-mouse PD-L1 antibody similar to the anti-human PD-L1 antibodies durvalumab and atezolizumab. Consistent with clinical observations for ICB in melanoma, anti-PD-L1 treatment elicited complete and durable response in a subset of melanoma-bearing mice. We also observed tumor growth delay or regression followed by recurrence. For early treatment assessment, we analyzed gene expression profiles, T-cell infiltration, and T-cell receptor (TCR) signatures in regressing tumors compared with tumors exhibiting no response to anti-PD-L1 treatment. We found that CD8+ T-cell tumor infiltration corresponded to response to treatment, and that anti-PD-L1 gene signature response indicated an increase in antigen processing and presentation, cytokine-cytokine receptor interaction, and natural killer cell-mediated cytotoxicity. TCR sequence data suggest that an anti-PD-L1-mediated melanoma regression response requires not only an expansion of the TCR repertoire that is unique to individual mice, but also tumor access to the appropriate TCRs. Thus, this melanoma model recapitulated the variable response to ICB observed in patients and exhibited biomarkers that differentiate between early response and resistance to treatment, providing a valuable platform for prediction of successful immunotherapy. IMPLICATIONS: Our melanoma model recapitulates the variable response to anti-PD-L1 observed in patients and exhibits biomarkers that characterize early antibody response, including expansion of the TCR repertoire.

Multimodel preclinical platform predicts clinical response of melanoma to immunotherapy.

Although immunotherapy has revolutionized cancer treatment, only a subset of patients demonstrate durable clinical benefit. Definitive predictive biomarkers and targets to overcome resistance remain unidentified, underscoring the urgency to develop reliable immunocompetent models for mechanistic assessment. Here we characterize a panel of syngeneic mouse models, representing a variety of molecular and phenotypic subtypes of human melanomas and exhibiting their diverse range of responses to immune checkpoint blockade (ICB). Comparative analysis of genomic, transcriptomic and tumor-infiltrating immune cell profiles demonstrated alignment with clinical observations and validated the correlation of T cell dysfunction and exclusion programs with resistance. Notably, genome-wide expression analysis uncovered a melanocytic plasticity signature predictive of patient outcome in response to ICB, suggesting that the multipotency and differentiation status of melanoma can determine ICB benefit. Our comparative preclinical platform recapitulates melanoma clinical behavior and can be employed to identify mechanisms and treatment strategies to improve patient care.

The Indenoisoquinoline TOP1 Inhibitors Selectively Target Homologous Recombination-Deficient and Schlafen 11-Positive Cancer Cells and Synergize with Olaparib.

PURPOSE: Irinotecan and topotecan are used to treat a variety of different cancers. However, they have limitations, including chemical instability and severe side effects. To overcome these limitations, we developed the clinical indenoisoquinolines: LMP400 (indotecan), LMP776 (indimitecan), and LMP744. The purpose of the study is to build the molecular rationale for phase II clinical trials. EXPERIMENTAL DESIGN: CellMinerCDB (http://discover.nci.nih.gov/cellminercdb) was used to mine the cancer cell lines genomic databases. The causality of Schlafen11 (SLFN11) was validated in isogenic cell lines. Because topoisomerase I (TOP1)-mediated replication DNA damage is repaired by homologous recombination (HR), we tested the "synthetic lethality" of HR-deficient (HRD) cells. Survival and cell-cycle alterations were performed after drug treatments in isogenic DT40, DLD1, and OVCAR cell lines with BRCA1, BRCA2, or PALB2 deficiencies and in organoids cultured from prostate cancer patient-derived xenografts with BRCA2 loss. We also used an ovarian orthotopic allograft model with BRCA1 loss to validate the efficacy of LMP400 and olaparib combination. RESULTS: CellMinerCDB reveals that SLFN11, which kills cells undergoing replicative stress, is a dominant drug determinant to the clinical indenoisoquinolines. In addition, BRCA1-, BRCA2-, and PALB2-deficient cells were hypersensitive to the indenoisoquinolines. All 3 clinical indenoisoquinolines were also synergistic with olaparib, especially in the HRD cells. The synergy between LMP400 and olaparib was confirmed in the orthotopic allograft model harboring BRCA1 loss. CONCLUSIONS: Our results provide a rationale for molecularly designed clinical trials with the indenoisoquinolines as single agents and in combination with PARP inhibitors in HRD cancers expressing SLFN11.

Synergistic targeted inhibition of MEK and dual PI3K/mTOR diminishes viability and inhibits tumor growth of canine melanoma underscoring its utility as a preclinical model for human mucosal melanoma.

Human mucosal melanoma (MM), an uncommon, aggressive and diverse subtype, shares characteristics with spontaneous MM in dogs. Although BRAF and N-RAS mutations are uncommon in MM in both species, the majority of human and canine MM evaluated exhibited RAS/ERK and/or PI3K/mTOR signaling pathway activation. Canine MM cell lines, with varying ERK and AKT/mTOR activation levels reflective of naturally occurring differences in dogs, were sensitive to the MEK inhibitor GSK1120212 and dual PI3K/mTOR inhibitor NVP-BEZ235. The two-drug combination synergistically decreased cell survival in association with caspase 3/7 activation, as well as altered expression of cell cycle regulatory proteins and Bcl-2 family proteins. In combination, the two drugs targeted their respective signaling pathways, potentiating reduction of pathway mediators p-ERK, p-AKT, p-S6, and 4E-BP1 in vitro, and in association with significantly inhibited solid tumor growth in MM xenografts in mice. These findings provide evidence of synergistic therapeutic efficacy when simultaneously targeting multiple mediators in melanoma with Ras/ERK and PI3K/mTOR pathway activation.

A preclinical orthotopic model for glioblastoma recapitulates key features of human tumors and demonstrates sensitivity to a combination of MEK and PI3K pathway inhibitors.

Current therapies for glioblastoma multiforme (GBM), the highest grade malignant brain tumor, are mostly ineffective, and better preclinical model systems are needed to increase the successful translation of drug discovery efforts into the clinic. Previous work describes a genetically engineered mouse (GEM) model that contains perturbations in the most frequently dysregulated networks in GBM (driven by RB, KRAS and/or PI3K signaling and PTEN) that induce development of Grade IV astrocytoma with properties of the human disease. Here, we developed and characterized an orthotopic mouse model derived from the GEM that retains the features of the GEM model in an immunocompetent background; however, this model is also tractable and efficient for preclinical evaluation of candidate therapeutic regimens. Orthotopic brain tumors are highly proliferative, invasive and vascular, and express histology markers characteristic of human GBM. Primary tumor cells were examined for sensitivity to chemotherapeutics and targeted drugs. PI3K and MAPK pathway inhibitors, when used as single agents, inhibited cell proliferation but did not result in significant apoptosis. However, in combination, these inhibitors resulted in a substantial increase in cell death. Moreover, these findings translated into the in vivo orthotopic model: PI3K or MAPK inhibitor treatment regimens resulted in incomplete pathway suppression and feedback loops, whereas dual treatment delayed tumor growth through increased apoptosis and decreased tumor cell proliferation. Analysis of downstream pathway components revealed a cooperative effect on target downregulation. These concordant results, together with the morphologic similarities to the human GBM disease characteristics of the model, validate it as a new platform for the evaluation of GBM treatment.

"Glowing head" mice: a genetic tool enabling reliable preclinical image-based evaluation of cancers in immunocompetent allografts.

Preclinical therapeutic assessment currently relies on the growth response of established human cell lines xenografted into immunocompromised mice, a strategy that is generally not predictive of clinical outcomes. Immunocompetent genetically engineered mouse (GEM)-derived tumor allograft models offer highly tractable preclinical alternatives and facilitate analysis of clinically promising immunomodulatory agents. Imageable reporters are essential for accurately tracking tumor growth and response, particularly for metastases. Unfortunately, reporters such as luciferase and GFP are foreign antigens in immunocompetent mice, potentially hindering tumor growth and confounding therapeutic responses. Here we assessed the value of reporter-tolerized GEMs as allograft recipients by targeting minimal expression of a luciferase-GFP fusion reporter to the anterior pituitary gland (dubbed the "Glowing Head" or GH mouse). The luciferase-GFP reporter expressed in tumor cells induced adverse immune responses in wildtype mouse, but not in GH mouse, as transplantation hosts. The antigenicity of optical reporters resulted in a decrease in both the growth and metastatic potential of the labeled tumor in wildtype mice as compared to the GH mice. Moreover, reporter expression can also alter the tumor response to chemotherapy or targeted therapy in a context-dependent manner. Thus the GH mice and experimental approaches vetted herein provide concept validation and a strategy for effective, reproducible preclinical evaluation of growth and response kinetics for traceable tumors.

Pathway-specific engineered mouse allograft models functionally recapitulate human serous epithelial ovarian cancer.

The high mortality rate from ovarian cancers can be attributed to late-stage diagnosis and lack of effective treatment. Despite enormous effort to develop better targeted therapies, platinum-based chemotherapy still remains the standard of care for ovarian cancer patients, and resistance occurs at a high rate. One of the rate limiting factors for translation of new drug discoveries into clinical treatments has been the lack of suitable preclinical cancer models with high predictive value. We previously generated genetically engineered mouse (GEM) models based on perturbation of Tp53 and Rb with or without Brca1 or Brca2 that develop serous epithelial ovarian cancer (SEOC) closely resembling the human disease on histologic and molecular levels. Here, we describe an adaptation of these GEM models to orthotopic allografts that uniformly develop tumors with short latency and are ideally suited for routine preclinical studies. Ovarian tumors deficient in Brca1 respond to treatment with cisplatin and olaparib, a PARP inhibitor, whereas Brca1-wild type tumors are non-responsive to treatment, recapitulating the relative sensitivities observed in patients. These mouse models provide the opportunity for evaluation of effective therapeutics, including prediction of differential responses in Brca1-wild type and Brca1-deficient tumors and development of relevant biomarkers.