Lung adenocarcinoma (LUAD) shows extensive lineage plasticity and early metastatic dissemination, but the oncogenic events that actively drive these processes remain poorly defined. Here, we identify coordinated PRKCI and ECT2 copy number gain, present in approximately 30% of human LUADs, as a driver of developmental reprogramming and metastasis. Using a genetically engineered mouse model that recapitulates PRKCI-ECT2 gain in Kras/Trp53-driven LUAD, we show that elevated PKCι-ECT2 signaling rewires tumor trajectory in a cell-of-origin-dependent manner. Alveolar type II cell-derived tumors dedifferentiate into a distal SOX9high progenitor-like state associated with aggressive growth and liver metastasis, whereas club cell-derived tumors transition into a foregut SOX2high progenitor-like state that supports lineage infidelity and histological transformation. Human LUAD analyses support these progenitor programs as clinically relevant features of PRKCI-ECT2 gain.

Protein kinase C (PKC) isozymes are ubiquitous kinases that direct diverse cellular pathways and are important drug targets for the treatment of cancer and neurological diseases. PKCs are auto-regulating enzymes governed by phospholipid and Ca2+ signals via a mechanism that has remained enigmatic due to a paucity of structural information. Herein we present a series of structures of the full-length human PKCβI and PKCβII isozymes. These structures reveal the molecular basis by which PKCs maintain an auto-inhibited state, convert to a defined and ordered active conformation via a "lipid-lever" mechanism of allosteric activation, and how isoform-specific differences alter their allosteric regulatory mechanisms. We show that endoxifen, a recently identified PKCβI inhibitor, can alter the allosteric regulatory mechanism of PKCβI, providing a proof of concept for allosteric regulators of PKCs. Collectively, our data describe a foundational molecular model of second messenger-mediated allosteric regulation of PKCs that underpins PKC function, misregulation, and mechanisms of inhibition.

Lung adenocarcinoma (LUAD), the most prevalent form of lung cancer, is characterized by aggressive growth, immune resistance, and high tumor heterogeneity. Here, we demonstrate that genetic loss of protein kinase Cι (PKCι), which is found in ∼20% of LUAD patients, alters the trajectory of mouse Kras/Trp53-driven LUAD tumors from one resembling lung development to one mimicking lung regeneration. As a result, a major subset of tumor cells with PKCι loss exhibit cellular senescence and transcriptional similarities to the pre-AT1 transitional cell state (PATS) observed during alveolar regeneration after lung injury. Senescent PATS-like tumor cells inhibit cellular plasticity through stable proliferation arrest and induce formation of tertiary lymphoid structures (TLSs) that enhance anti-tumor immunity and patient responses to immune therapies. Importantly, human LUADs harboring genetic PKCι loss also contain TLSs and PATS-like tumor cells. Therefore, PKCι expression in mouse and human LUAD dictates tumor trajectory, cellular plasticity, and the immune microenvironment.

OBJECTIVES:This trial served as a proof-of-concept for whether inhibition of protein kinase C iota (PKCι) with auranofin and sirolimus provide antineoplastic effects in patients with recurrent high-grade serous ovarian cancer. METHODS:This drug combination was administered to patients with recurrent high-grade serous ovarian cancer. Dosing was based on unpublished phase 1 data and consisted of auranofin 6 mg and sirolimus 5 mg both orally per day of a 28-day cycle. The primary endpoint was tumor response. Available tumor tissue was assessed for PKCι protein expression by immunohistochemistry (IHC) and PRKCI copy number by fluorescence in-situ hybridization (FISH) after the start of cancer therapy. RESULTS:Twenty-two patients were enrolled, and 21 were evaluable for all clinical trial endpoints. One patient was unevaluable because she did not receive a full chemotherapy cycle. No tumor responses were seen in the first 21 patients, resulting in early trial termination per a priori trial design. The median progression-free survival was 2.1 months (95% CI: 1.8-3.7). The median overall survival was 4.4 months (95% CI: 2.6-12.5). Fourteen (67%) patients had at least one grade 3 or worse adverse event. Nineteen of 21 evaluable patients had available tumor tissue, which showed the median PKCι copy number averaged per cell of 3 (range: 2 to 7), and PKCι expression (at least 1+) in all. CONCLUSIONS:As prescribed here, auranofin and sirolimus manifested no antineoplastic activity in patients with recurrent high-grade serous ovarian cancer that expressed PKCι.

Abstract Lung squamous cell carcinoma (LUSC) is a devastating disease, accounting for ~30% of all lung cancer diagnoses and ~40,000 deaths annually in the United States alone. LUSC patients suffer from high prevalence of relapse and a dismal 5-year survival of only ~24%. The lack of animal models that reflect the salient features of human disease to assess the safety and efficacy of drugs, and to explore the underlying molecular mechanisms of LUSC tumor biology, is a major impediment for LUSC patients. We have identified concomitant 3q26 copy number gain (CNG) and TP53 loss as a defining genetic alteration present in ~90% of LUSC tumors. Furthermore, we have identified and characterized three 3q26 oncogenes (PRKCI, ECT2 and SOX2) that genetically and biochemically collaborate to drive LUSC tumor formation. Using syngeneic mouse modeling, we have demonstrated that coordinate overexpression of Prkci, Ect2 and Sox2, and loss of Trp53 in ex vivo cultures of lung basal stem cells (LBSCs; a cell of origin for LUSC) drives transformation of these cells into tumors of exclusively LUSC histopathology. These findings lead to the hypothesis that PRKCI, ECT2 and SOX2 represent a multigenic driver of 3q26 CNG-driven LUSC, which we term the 3q26 OncCassette. Based in these findings, we have developed and begun characterizing the first autochthonous genetically engineered mouse model (GEMM) to study 3q26 OncCassette-driven tumorigenesis. Our novel immune competent GEMM will provide critical insight into tumor initiation and progression of preneoplastic lesions to malignant LUSC and allow characterization of the immune landscape in these tumors. Citation Format: Kayleah M. Meneses, Yi Liu, Capella R. Weems, Lee Jamieson, Duy T. Nguyen, Verline Justilien, Nicole R. Murray, Alan P. Fields. Modeling the 3q26 OncCassette using genetically engineered mice [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 1434.

Abstract Lung cancer is the leading cause of cancer deaths in the US. Non-small cell lung cancer (NSCLC) accounts for ~80% of lung cancer cases and is further classified into adenocarcinoma (LUAD), squamous cell carcinoma (LUSC), and large cell carcinoma. New approaches targeting pathways in NSCLC have emerged, resulting in encouraging new treatments that benefit LUAD patients, but very few advances have been made in the treatment options for LUSC which accounts for ~30% of lung cancer cases. NSCLCs, including LUSCs, contain a sub-population of cells that exhibit properties of cancer stem cells (CSCs) and are responsible for tumor initiation, maintenance, therapeutic resistance, and relapse. Therefore, CSCs must be effectively targeted to elicit long lasting therapeutic results in LUSC patients. Our studies have shown that: 1) PKCι is an oncogene and prognostic marker in LUSC; 2) copy number gains (CNGs) of the PKCι gene (PRKCI) drives overexpression of PKCι in 90% of LUSCs; 3) PKCι phosphorylates SOX2, and controls SOX2-mediated transcriptional activation of Hedgehog Acyl Transferase, resulting in Hedgehog (Hh) pathway activation that drives a CSC phenotype in LUSC cells; and 4) the FDA approved compound Auranofin (ANF) is a potent inhibitor of oncogenic PKCι. In this study, we assessed the effect of ANF in combination with the Hh pathway inhibitor, LDE225, on LUSC CSCs and tumor growth. Cell viability, sphere formation and soft agar assays were used to assess the effects of ANF and LDE225 alone or in combination on the growth of LUSC CSCs with PRKCI CNGs or LUAD and LUSC CSCs without PRKCI CNG to determine if PRKCI CNG can be used to predict response to ANF and LDE225 and test the selectivity of response for LUSC. The effects of ANF and LDE225 on the levels of PKCι-Hh signaling components was assessed by qPCR and western blotting. Finally, the efficacy of ANF and LDE225 in combination was evaluated in human primary LUSC patient-derived xenograft (PDX) models that harbor PRKCI CNGs and the ability of PKCι-Hh signaling intermediates to serve as biomarkers of PKCι-Hh pathway inhibition was validated in vivo. We observed a dose-dependent decrease in cell proliferation, clonal expansion and transformed growth in CSCs treated with ANF and LDE225 alone. ANF and LDE225 combined treatment of LUSC CSCs with PRKCI CNGs (but not LUAD or LUSC CSCs without PRKCI CNG) synergistically inhibited CSC phenotypes based on results of combination index analysis. Furthermore, treatment of LUSC PDX models with ANF and LDE225 in combination had a synergistic effect on tumor growth inhibition. Lastly, ANF and LDE225 treatment led to inhibition of PKCι-Hh signaling intermediates demonstrating the on-target effects of treatment. Our data indicate that a PKCι-Hh vertical blockade strategy of combined ANF and LDE225 effectively targets LUSC CSCs and blocks tumor growth in vivo and provides compelling rationale to develop this novel combination strategy further in clinical trials. Citation Format: Dania Alqasrawi, Ryan Argo, Christine Ratliff Rang, Prita Pandya, Skyeler Klinge, Jennifer Lindemann, Nyla Searl, Alan Fields, Verline Justilien. A novel combination therapy for lung squamous cell carcinoma [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 5434.

Abstract Lung adenocarcinoma (LUAD), the most prevalent subtype of lung cancer, can arise from two different cell populations: alveolar type 2 (AT2) cells and bronchiolar club cells. By utilizing genomic data from the cancer genome atlas program, human LUAD cell lines, and the well-characterized LSL-KrasG12D/Trp53-/- (KP) mouse model, we demonstrated that tumors arising from club cells are much more aggressive and follow a different trajectory of tumorigenesis when compared to those arising from AT2 cells. Intriguingly, our data strongly suggest that expression and signaling activity of protein kinase C iota (PKCι) can profoundly affect the cell of origin and oncogenic signaling pathways employed by resultant tumors. Specifically, KP-mediated transformation of club cells appears to be dependent on PKCι-ELF3-NOTCH3 and PKCι-YAP1 signaling axes, while AT2 cell malignancy is dependent on the PKCι-independent WNT signaling. In addition, club-cell-origin tumors exhibit an enrichment of cancer stem cells and increased infiltration of immunosuppressive myeloid cells in comparison to AT2-cell-origin tumors. This results in accelerated progression and increased likelihood of metastasis by LUAD tumors that originate from club cells. Taken together, our results indicate that PKCι expression and signaling activity can profoundly influence the cells of origin, tumor trajectories, and therapeutic vulnerabilities of LUAD tumors. Citation Format: Duy T. Nguyen, Ning Yin, Capella R. Weems, Lee Jamieson, Kayleah Meneses, Yi Liu, Nicole R. Murray, Alan P. Fields. A lineage-specific role of oncogenic protein kinase Cι in lung adenocarcinoma [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 4299.

Supplementary Figure 5 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Supplementary Figure 1 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Supplementary Tables and Methods.

Supplementary Figure Legends 1-6 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

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Supplementary Figure 4 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Supplementary Figure 1 from Atypical Protein Kinase Cι Is Required for Bronchioalveolar Stem Cell Expansion and Lung Tumorigenesis

Lung squamous cell carcinoma (LUSC) is a major histological subtype of lung cancer accounting for ~30% of all lung cancer diagnoses. LUSC patients suffer from poor therapeutic response, high relapse rate, and poor prognosis. A major reason for this bleak outcome is the lack of well characterized oncogenic drivers of LUSC. This has stalled the generation of relevant genetic pre-clinical mouse models amenable to studying LUSC tumorigenesis in vivo and developing targeted therapeutics to treat LUSC patients. Our group has identified concomitant 3q26 copy number gain (CNG) and TP53 loss as defining genetic alterations present in ~90% of LUSC tumors. Furthermore, we have identified three 3q26 oncogenes (PRKCI, ECT2 and SOX2) that genetically and biochemically collaborate to drive LUSC tumor formation. Coordinate overexpression of Prkci, Ect2 and Sox2, and loss of Trp53 in ex vivo cultures of lung basal stem cells (LBSCs; a potential cell of origin for LUSC) is both necessary and sufficient to transform these cells into orthotopic LUSC tumors in syngeneic mice. These findings indicate that 3q26 amplification and TP53 loss play a critical role in the initiation and progression of LUSC. Based on these data, we have developed the first autochthonous genetically engineered mouse model (GEMM) to study the role of 3q26 CNG in LUSC tumorigenesis in vivo. Our GEMM allows inducible overexpression of Prkci, Ect2, and Sox2 and knockout of Trp53 (PES/Trp53−/−) in lung tissue. Tumorigenesis will be initiated in PES/Trp53−/− mice using an established protocol to target transgene recombination to LBSCs in vivo. These mice will be assessed for tumor latency, survival, tumor burden, and metastatic potential. In related studies, we used bioinformatics analyses on primary LUSC tumors from the Cancer Genome Atlas’ database to identify 13 genes in addition to PRKCI, ECT2 and SOX2, whose expression is driven by 3q26 amplification. These genes were functionally characterized by assessing the effect of shRNA-mediated gene silencing on soft-agar growth of human H520 LUSC cells, a cell line that harbors 3q26 amplification. Our results revealed that knockdown of each of these genes independently resulted in a significant decrease in soft agar colony formation. We further assessed these genes through RT-qPCR mRNA expression profiling comparing PES/Trp53−/- transformed LBSCs to non-transformed LBSCs. These genes were significantly upregulated in PES/Trp53−/- transformed LBSCs, suggesting that they play important roles in LBSC transformation. Future work will further assess the molecular mechanisms by which these additional 3q26 genes contribute to LUSC cell transformation and assess the role of alternate cells of origin (Club and AT2) in 3q26-driven LUSC tumor formation using our PES/Trp53−/− GEMM. Citation Format: Kayleah M. Meneses, Capella Weems, Yi Liu, Verline Justilien, Nicole R. Murray, Alan P. Fields. Characterizing 3q26 copy number gain in lung squamous cell carcinoma through a novel genetically engineered mouse model [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 27.

Supplementary Table 1 from Protein Kinase Cβ Is an Effective Target for Chemoprevention of Colon Cancer

Endoxifen, a secondary tamoxifen metabolite, is a potent antiestrogen exhibiting estrogen receptor alpha (ERα) binding at nanomolar concentrations. Phase I/II clinical trials identified clinical activity of Z-endoxifen (ENDX), in endocrine-refractory metastatic breast cancer as well as ERα+ solid tumors, raising the possibility that ENDX may have a second, ERα-independent, mechanism of action. An unbiased mass spectrometry approach revealed that ENDX concentrations achieved clinically with direct ENDX administration (5 µM), but not low concentrations observed during tamoxifen treatment (<0.1 µM), profoundly altered the phosphoproteome of the aromatase expressing MCF7AC1 cells with limited impact on the total proteome. Computational analysis revealed protein kinase C beta (PKCβ) and protein kinase B alpha or AKT1 as potential kinases responsible for mediating ENDX effects on protein phosphorylation. ENDX more potently inhibited PKCβ1 kinase activity compared to other PKC isoforms, and ENDX binding to PKCβ1 was confirmed using Surface Plasma Resonance. Under conditions that activated PKC/AKT signaling, ENDX induced PKCβ1 degradation, attenuated PKCβ1-activated AKTSer473 phosphorylation, diminished AKT substrate phosphorylation, and induced apoptosis. ENDX’s effects on AKT were phenocopied by siRNA-mediated PKCβ1 knockdown or treatment with the pan-AKT inhibitor, MK-2206, while overexpression of constitutively active AKT diminished ENDX-induced apoptosis. These findings, which identify PKCβ1 as an ENDX target, indicate that PKCβ1/ENDX interactions suppress AKT signaling and induce apoptosis in breast cancer.

Supplementary Tables 1-2 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Supplementary Figure 1 from Atypical Protein Kinase Cι Expression and Aurothiomalate Sensitivity in Human Lung Cancer Cells

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