Biology subjects

Kleczko, E. K.

Publications and source records attributed to Kleczko, E. K..

5 recordsLinked to original sources

bioRxiv2022

Novel EGFR-Mutant Mouse Models of Lung Adenocarcinoma Reveal Adaptive Immunity Requirement for Durable Osimertinib Response

Lung cancers bearing oncogenically-mutated EGFR represent a significant fraction of lung adenocarcinomas (LUADs) for which EGFR-targeting tyrosine kinase inhibitors (TKIs) provide a highly effective therapeutic approach. However, these lung cancers eventually acquire resistance and undergo progression within a characteristically broad treatment duration range. Our previous study of EGFR mutant lung cancer biopsies highlighted the positive association of a TKI-induced interferon {gamma} transcriptional response with increased time to treatment progression. To test the hypothesis that host immunity contributes to the TKI response, we developed novel genetically-engineered mouse models of EGFR mutant lung cancer bearing exon 19 deletions (del19) or the L860R missense mutation. Both oncogenic EGFR mouse models developed multifocal LUADs from which transplantable cancer cell lines sensitive to the EGFR-specific TKIs, gefitinib and osimertinib, were derived. When propagated orthotopically in the left lungs of syngeneic C57BL/6 mice, deep and durable shrinkage of the cell line-derived tumors was observed in response to daily treatment with osimertinib. By contrast, orthotopic tumors propagated in immune deficient nu/nu mice exhibited modest tumor shrinkage followed by rapid progression on continuous osimertinib treatment. Importantly, osimertinib treatment significantly increased intratumoral CD3+ T cell content relative to diluent treatment. The findings provide strong evidence supporting the requirement for adaptive immunity in the durable therapeutic control of EGFR mutant lung cancer.

cancer biology↗

bioRxiv2022

Evaluation of KRASG12C Inhibitor Responses in Novel Murine KRASG12C Lung Cancer Cell Line Models

The KRAS(G12C) mutation is the most common genetic mutation in North American lung adenocarcinoma patients. Recently, direct inhibitors of the KRASG12C protein have been developed and demonstrate clinical response rates of 37-43%. Importantly, these agents fail to generate durable therapeutic responses with median progression-free survival of ~6.5 months. To provide models for further preclinical improvement of these inhibitors, we generated three novel murine KRASG12C-driven lung cancer cell lines. The co-occurring NRASQ61L mutation in KRASG12C-positive LLC cells was deleted and the KRASG12V allele in CMT167 cells was edited to KRASG12C with CRISPR/Cas9 methods. Also, a novel murine KRASG12C line, mKRC.1, was established from a tumor generated in a genetically-engineered mouse model. The three lines exhibit similar in vitro sensitivities to KRASG12C inhibitors (MRTX-1257, AMG-510), but distinct in vivo responses to MRTX-849 ranging from progressive growth with orthotopic LLC-NRAS KO tumors to marked shrinkage with mKRC.1 tumors. All three cell lines exhibited synergistic in vitro growth inhibition with MRTX-1257 and the SHP2 inhibitor, RMC-4550 and the MRTX-849/RMC-4550 combination yielded tumor shrinkage in orthotopic LLC-NRAS KO tumors propagated in syngeneic mice. Notably, this synergistic combination response was lost in athymic nu/nu mice, supporting a growing literature demonstrating a role for adaptive immunity in the response to this class of drugs. These new models of murine KRASG12C mutant lung cancer should prove valuable for identifying improved therapeutic combination strategies with KRASG12C inhibitors. Contribution to the Field StatementThe development of KRASG12C inhibitors has not impacted treatment of lung cancers bearing the KRASG12C mutation to the degree that tyrosine kinase inhibitors have changed the treatment outcomes for patients bearing oncogenic mutations in receptor tyrosine kinases. Thus, the field is now exploring combination strategies with KRASG12C inhibitors that may enhance their clinical benefit. Moreover, published findings indicate that host immunity contributes to efficacy of oncogene-directed inhibitors including KRASG12C inhibitors. Thus, these novel murine KRASG12C-driven lung cancer cell lines will provide valuable models for preclinical evaluation of novel drug combinations in immune competent hosts.

cancer biology↗

bioRxiv2022

The Tryptophan Metabolizing Enzyme Indoleamine 2,3-Dioxygenase 1 Regulates Polycystic Kidney Disease Progression

Autosomal dominant polycystic kidney disease (ADPKD), the most common monogenic nephropathy, is characterized by phenotypic variability exceeding genic effects. Dysregulated metabolism and immune cell function are key disease modulators. The tryptophan metabolites, kynurenines, produced through IDO1, are known immunomodulators. Here, we study the role of tryptophan metabolism in PKD using an orthologous disease model (C57Bl/6J Pkd1RC/RC). We found elevated kynurenine and IDO1 levels in Pkd1RC/RC kidneys versus wildtype. Further, IDO1 levels were increased in ADPKD cell lines and patient cyst cells. Genetic Ido1 loss in Pkd1RC/RC animals resulted in reduced PKD severity as measured by %kidney weight/body weight and cystic index. Consistent with a immunomodulatory role of kynurenines, Pkd1RC/RC;Ido1-/- mice presented with significant changes in the cystic immune microenvironment (CME) versus controls. Of note, kidney macrophage numbers decreased and CD8+ T cell numbers increased, both known PKD modulators. Also, pharmacological IDO1 inhibition using a tryptophan analog in Pkd1RC/RC animals resulted in less severe PKD versus controls with similar changes in the CME as in the genetic model. Together, our data suggest that tryptophan metabolism is dysregulated in ADPKD and that its inhibition results in changes to the CME and slows disease progression, making IDO1 a novel therapeutic target for ADPKD.

molecular biology↗

bioRxiv2022

Immune Checkpoint Activity Regulates Polycystic Kidney Disease Progression

Innate and adaptive immune cells modulate Autosomal Dominant Polycystic Kidney Disease (ADPKD) severity, a common kidney disease with inadequate treatment options. ADPKD shares parallels with cancer where immune checkpoint inhibitors have been shown to reactivate CD8+ T cells and slow tumor growth. We have shown that, in PKD, CD8+ T cell loss worsens disease. This study used orthologous early-onset and adult-onset ADPKD models (Pkd1 p.R3277C) to evaluate the role of immune checkpoints in PKD. Flow cytometry of kidney cells showed increased levels of PD-1 on CD8+ T cells and PD-L1 on macrophages and epithelial cells in Pkd1RC/RC mice versus wildtypes, paralleling disease severity. PD-L1 was also upregulated in ADPKD human cells and patient kidney tissue versus controls. Genetic PD-L1 loss or treatment with an anti-PD-1 antibody did not impact PKD severity in early-onset or adult-onset ADPKD models. However, treatment with anti-PD-1 plus anti-CTLA-4, blocking two immune checkpoints, improved PKD outcomes in adult-onset ADPKD mice; neither monotherapy altered PKD. Combination therapy resulted in increased kidney CD8+ T cell numbers/activation and decreased kidney regulatory T cell numbers. Together, our data suggests that immune checkpoint activation is an important feature of and potential novel therapeutic target in ADPKD.

immunology↗

bioRxiv2022

Adaptive immunity is required for durable responses to alectinib in murine models of EML4-ALK lung cancer

PurposeLung cancers bearing oncogenic EML4-ALK fusions respond to targeted tyrosine kinase inhibitors (TKIs; e.g. alectinib), with variation in the degree of shrinkage and duration of treatment (DOT). We previously demonstrated a positive association of a TKI-induced interferon gamma (IFN{gamma}) transcriptional response with DOT in EGFR-mutant lung cancers. Herein, we used murine models of EML4-ALK lung cancer to test a role for host immunity in the therapeutic response to alectinib. Experimental DesignThree murine EML4-ALK cell lines (EA1, EA2, EA3) were implanted orthotopically into the lungs of immunocompetent and immunodeficient mice and treated with alectinib. Tumor volumes were serially measured by CT. Immune cell content was measured by flow cytometry, multispectral immunofluorescence and CyTOF. Transcriptional responses to alectinib were assessed by RNAseq and secreted chemokines were measured by ELISA. ResultsAll cell lines were sensitive to alectinib in vitro. EA1 and EA3 tumors retained residual disease that rapidly progressed upon termination of treatment while EA2 tumors were eliminated by TKI treatment. Alectinib induced inflammatory transcriptional programs and multiple chemokines in all cell lines while untreated tumors exhibited distinct baseline chemokine expression patterns and content of CD8+ T cells and myeloid subsets. When propagated in immune-deficient mice, all three cell line-derived lung tumor models exhibited significant shrinkage followed by prompt progression despite continuous alectinib treatment. ConclusionsThe findings support an hypothesis that host and TKI-stimulated production of chemokines by tumor cells promotes functional engagement of adaptive immune cells within the tumor microenvironment that enhances the durability and depth of TKI response. Statement of Translational RelevancePatients with metastatic lung cancer harboring ALK fusions are treated with targeted tyrosine kinase inhibitors (TKI) in the first line setting. Despite bearing the same driver oncogene, patients experience a range of tumor burden reduction and variable amounts of residual disease. Residual disease burden associates with patient survival and contributes to the emergence of drug resistance yielding treatment failure. The factors mediating this differential response to TKI and residual disease are incompletely understood. Our group has developed a panel of murine ALK driven lung cancer cell lines that reproducibly show differences in the depth and duration of response when implanted into immunocompetent mice. Data using this model indicate that the presence of CD8+ T cells is a major contributor to the depth and duration of response. These models will be critical in developing rational combination therapies to augment the immune microenvironment engagement along with TKIs to improve outcomes for these patients.

cancer biology↗