PI3Kß inhibition enhances ALK-inhibitor sensitivity in ALK-rearranged lung cancer.

Treatment with anaplastic lymphoma kinase (ALK) inhibitors significantly improves outcome for non-small-cell lung cancer (NSCLC) patients with ALK-rearranged tumors. However, clinical resistance typically develops over time and, in the majority of cases, resistance mechanisms are ALK-independent. We generated tumor cell cultures from multiple regions of an ALK-rearranged clinical tumor specimen and deployed functional drug screens to identify modulators of ALK-inhibitor response. This identified a role for PI3Kß and EGFR inhibition in sensitizing the response regulating resistance to ALK inhibition. Inhibition of ALK elicited activation of EGFR, and subsequent MAPK and PI3K-AKT pathway reactivation. Sensitivity to ALK targeting was enhanced by inhibition or knockdown of PI3Kß. In ALK-rearranged primary cultures, the combined inhibition of ALK and PI3Kß prevented the EGFR-mediated ALK-inhibitor resistance, and selectively targeted the cancer cells. The combinatorial effect was seen also in the background of TP53 mutations and in epithelial-to-mesenchymal transformed cells. In conclusion, combinatorial ALK- and PI3Kß-inhibitor treatment carries promise as a treatment for ALK-rearranged NSCLC.

Development of an adenosquamous carcinoma histopathology - selective lung metastasis model.

Preclinical tumor models with native tissue microenvironments provide essential tools to understand how heterogeneous tumor phenotypes relate to drug response. Here we present syngeneic graft models of aggressive, metastasis-prone histopathology-specific NSCLC tumor types driven by KRAS mutation and loss of LKB1 (KL): adenosquamous carcinoma (ASC) and adenocarcinoma (AC). We show that subcutaneous injection of primary KL; ASC cells results in squamous cell carcinoma (SCC) tumors with high levels of stromal infiltrates, lacking the source heterogeneous histotype. Despite forming subcutaneous tumors, intravenously injected KL;AC cells were unable to form lung tumors. In contrast, intravenous injection of KL;ASC cells leads to their lung re-colonization and lesions recapitulating the mixed AC and SCC histopathology, tumor immune suppressive microenvironment and oncogenic signaling profile of source tumors, demonstrating histopathology-selective phenotypic dominance over genetic drivers. Pan-ERBB inhibition increased survival, while selective ERBB1/EGFR inhibition did not, suggesting a role of the ERBB network crosstalk in resistance to ERBB1/EGFR. This immunocompetent NSCLC lung colonization model hence phenocopies key properties of the metastasis-prone ASC histopathology, and serves as a preclinical model to dissect therapy responses and metastasis-associated processes.

Protocol to utilize fresh uncultured human lung tumor cells for personalized functional diagnostics.

Drug sensitivity data acquired from solid tumor-derived cultures are often unsuitable for personalized treatment guidance due to the lengthy turnaround time. Here, we present a protocol for determining ex vivo drug sensitivities using fresh uncultured human lung tumor-derived EpCAM+ epithelial cells (FUTCs). We describe steps for drug testing in FUTCs to identify tumor cell-selective single or combination therapy in 72 h of sample processing. The FUTC-based approach can also be used to predict in vivo resistance to known targeted therapies. For complete details on the use and execution of this protocol, please refer to Talwelkar et al. (2021).

Functional diagnostics using fresh uncultured lung tumor cells to guide personalized treatments.

Functional profiling of a cancer patient's tumor cells holds potential to tailor personalized cancer treatment. Here, we report the utility of fresh uncultured tumor-derived EpCAM+ epithelial cells (FUTCs) for ex vivo drug-response interrogation. Analysis of murine Kras mutant FUTCs demonstrates pharmacological and adaptive signaling profiles comparable to subtype-matched cultured cells. By applying FUTC profiling on non-small-cell lung cancer patient samples, we report robust drug-response data in 19 of 20 cases, with cells exhibiting targeted drug sensitivities corresponding to their oncogenic drivers. In one of these cases, an EGFR mutant lung adenocarcinoma patient refractory to osimertinib, FUTC profiling is used to guide compassionate treatment. FUTC profiling identifies selective sensitivity to disulfiram and the combination of carboplatin plus etoposide, and the patient receives substantial clinical benefit from treatment with these agents. We conclude that FUTC profiling provides a robust, rapid, and actionable assessment of personalized cancer treatment options.

SOX9 has distinct roles in the formation and progression of different non-small cell lung cancer histotypes.

The transcription factor SOX9 is a key regulator of multiple developmental processes and is frequently re-expressed in non-small cell lung cancer (NSCLC). Its precise role in the progression of NSCLC histotypes has, however, remained elusive. We show that SOX9 expression relates to poor overall survival and invasive histopathology in human non-mucinous adenocarcinoma and is absent in murine early minimally invasive and low in human in situ adenocarcinoma. Interestingly, despite wide SOX9 expression across advanced NSCLC histotypes, its genetic deletion in the murine KrasG12D ;Lkb1fl/fl model selectively disrupted only the growth of papillary NSCLC, without affecting the initiation of precursor lesions or growth of mucinous or squamous tissue. Spatial tissue phenotyping indicated a requirement of SOX9 expression for the progression of surfactant protein C-expressing progenitor cells, which gave rise to papillary tumours. Intriguingly, while SOX9 expression was dispensable for squamous tissue formation, its loss in fact led to enhanced squamous tumour metastasis, which was associated with altered collagen IV deposition in the basement membrane. Our work therefore demonstrates histopathology-selective roles for SOX9 in NSCLC progression, namely as a promoter for papillary adenocarcinoma progression, but an opposing metastasis-suppressing role in squamous histotype tissue. This attests to a pleiotropic SOX9 function, linked to the cell of origin and microenvironmental tissue contexts. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Patient-tailored design for selective co-inhibition of leukemic cell subpopulations.

The extensive drug resistance requires rational approaches to design personalized combinatorial treatments that exploit patient-specific therapeutic vulnerabilities to selectively target disease-driving cell subpopulations. To solve the combinatorial explosion challenge, we implemented an effective machine learning approach that prioritizes patient-customized drug combinations with a desired synergy-efficacy-toxicity balance by combining single-cell RNA sequencing with ex vivo single-agent testing in scarce patient-derived primary cells. When applied to two diagnostic and two refractory acute myeloid leukemia (AML) patient cases, each with a different genetic background, we accurately predicted patient-specific combinations that not only resulted in synergistic cancer cell co-inhibition but also were capable of targeting specific AML cell subpopulations that emerge in differing stages of disease pathogenesis or treatment regimens. Our functional precision oncology approach provides an unbiased means for systematic identification of personalized combinatorial regimens that selectively co-inhibit leukemic cells while avoiding inhibition of nonmalignant cells, thereby increasing their likelihood for clinical translation.