KP Tumors Research Articles

Abstract 3531: YAP1-mediated circadian oscillation in sarcoma

Abstract Soft tissue sarcomas have presented a unique challenge to researchers due to their heterogeneity. One common mechanism underlying more than 25% of diagnosed sarcomas is the deregulation of the Hippo pathway, a critical signaling cascade that responds to cell contact inhibition, among other factors, and negatively regulates cell proliferation. In quiescent cells the pathway remains “on,” which maintains activation of a kinase cascade that phosphorylates and degrades YAP1. However, inactivation of the pathway stabilizes YAP1 allowing it to translocate to the nucleus and activate transcription of pro-proliferative gene targets. Microarray analysis of muscle tissue from the LSL-KrasG12D/+;Trp53fl/fl (KP) mouse model of undifferentiated pleomorphic sarcoma (UPS) revealed that YAP1 deletion (KPY) reduces cell proliferation and increases expression of circadian rhythm genes including PER1. UPS is a commonly diagnosed and aggressive type of muscle-derived sarcoma. The KP model recapitulates human UPS morphologically and histologically, as well as by gene expression profiling. Although PER1 has primarily been characterized as a negative regulator of the circadian clock, upregulation of PER1 is known to modulate the G2/M cell cycle checkpoint at both the protein and mRNA level independent of p53. However, our findings represent a novel link between the circadian clock and the hippo pathway. Notably, deletion of YAP1 in KP tumors leads to a statistically significant 2.5 fold increase in expression of PER1. The study has two specific aims: 1) to identify the YAP1-dependent function of PER1 in sarcoma and 2) determine whether YAP1 directly or indirectly regulates PER1. We have validated the microarray results in KP tumor derived cell lines as well as in KP and KPY tumor tissue. I have also confirmed PER1 suppression in KP cells under YAP1 short hairpin RNA conditions. Our lab has previously observed that treatment with epigenetic modulating drugs JQ1, a BET inhibitor, and SAHA, an HDAC inhibitor, significantly decreased sarcoma growth in vivo and in vitro when administered alone and had an additive effect when combined. These effects can be explained in part by the finding that treatment with these inhibitors significantly reduces YAP1 expression. Under these conditions, as well as YAP1 knockdown conditions, I demonstrated via qRT-PCR and western blot that PER1 is significantly increased at both the protein and transcriptional levels in KP cells. Additionally, preliminary evidence from an MTT proliferation assay showed loss of PER1 increased sarcoma cell proliferation. Further supporting the hypothesis that PER1 modulation impacts sarcoma proliferation, it has been reported that MyoD, the master regulator of muscle cell differentiation, is itself a clock-controlled gene. Together, these results suggest that YAP1 represses PER1 expression in sarcoma, and that epigenetic treatments can cause re-expression of PER1 which functions to inhibit cell proliferation and may promote differentiation. Citation Format: Gloria Marino, Shuai Ye, Koreana Pak, Jennifer Shah, Jason Godfrey, Susan Chor, Shaun Egolf, T.S. Karin Eisinger-Mathason. YAP1-mediated circadian oscillation in sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3531. doi:10.1158/1538-7445.AM2017-3531

Abstract 2964: Stromal Hedgehog pathway activation suppresses growth and metastases of lung adenocarcinoma

Abstract Aberrant activation of the Hedgehog (Hh) signaling pathway, a crucial developmental pathway, drives the tumor growth of Gorlin-type cancers. However, recent data suggest that paracrine activation of the pathway is tumor suppressive rather than oncogenic in sporadic epithelial cancers. The role of the pathway in non-small lung cancer is poorly understood. Thus, we explored the role of stromal Hh pathway activation in growth of lung tumor epithelia. Human lung adenocarcinoma cell lines were used to probe SHH mRNA and protein expression. Co-culture of high SHH expressing cell lines with murine embryonic and lung fibroblasts were used to confirm and probe the role of paracrine SHH expression on the growth of lung cancer cells. The in vivo role of paracrine SHH was tested using autochthonous lung cancer models with conditional KRASG12Dactivation, p53 loss,, and SHH loss compared to wild-type SHH. In human lung adenocarcinoma patients, higher expression of SHH mRNA in lung adenocarcinoma correlated with poor overall and progression free survival. A scan of 35 human lung adenocarcinoma cell lines revealed heterogeneous expression of SHH and IHH with high expression found predominantly in mutant K-Ras lines. Co-culture of high SHH expressing tumor epithelial cells and Shh-Light2 reporter cell lines demonstrated that SHH activated the fibroblast reporter in a paracrine manner, rather than an autocrine effect on cancer cells. Treatment with the SMO inhibitor, KAAD-cyclopamine, also inhibited the growth of tumor epithelial cells in co-culture with NIH-3T3 fibroblast cells but the effect was decreased when co-cultured with lung fibroblasts. Genetic loss of SHH in an autochthonous mouse model, LSL-KrasG12D/+;Trp53fl/fl; Shhfl/fl (KPS) did not affect overall survival compared to LSL-KrasG12D/+;Trp53fl/fl (KP) mice However, early inhibition of the Hh pathway by anti-SHH/IHH antibody, 5E1, on KP mice resulted in significantly worse survival rates with increased metastatic burden compared to IgG treatment. Analysis of KP tumors revealed unexpected high levels of IHH mRNA by in situ hybridization that may account for the survival differences seen between genetic ablation and pharmaceutical inhibition of the Hh ligands. In conclusion, the Hh signaling pathway acts upon lung stromal cells in a paracrine fashion. Inhibition of Hh activity in vivo worsened mortality rate due to increase in tumor growth and metastases. Furthermore, mutant Kras lung cancers express high levels of IHH that dominates the tumor suppressive effect of our mouse models. Citation Format: Sahba Kasiri, Baozhi Chen, Alexandra Wilson, Umaru Barrie, Ummay Marriam, Zhiqun Zeng, Luc Girard, James Kim. Stromal Hedgehog pathway activation suppresses growth and metastases of lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2964. doi:10.1158/1538-7445.AM2017-2964

STK11/LKB1 co-mutations to predict for de novo resistance to PD-1/PD-L1 axis blockade in KRAS-mutant lung adenocarcinoma.

9016 Background: Identification of molecular predictors of response to PD-1/PD-L1 inhibitors is critical in order to maximize their therapeutic potential. We previously reported that KRAS-mutant lung adenocarcinomas (LUAC) with co-occurring genetic events in STK11/LKB1(KL) or TP53 (KP) define subgroups with marked differences in immune contexture, including a paucity of CD8+ TILs in KL LUAC. Here, we assess clinical responses to PD-1/PD-L1 therapy in KL and KP subsets, with data assembled under the auspices of the SU2C/ACS Lung Cancer Dream Team. Methods: Patients (pts) with metastatic KRAS-mutant LUAC who received at least one cycle of PD-1/PD-L1 therapy, were alive for ≥14 days thereafter, and had available molecular profiling were identified retrospectively. Efficacy assessment was based on RECIST v1.1. PD-L1 expression was tested using 22C3 pharmDx or E1L3N IHC assays and quantified as percent of staining tumor cell membranes. Isogenic derivatives of the LKR10 KrasLA1/+ murine LUAC cell line with CRISPR/Cas9-mediated Lkb1knockout were used in preclinical experiments. Results: 165 pts with KRAS-mutant LUAC who received PD-1/PD-L1 therapy were included [KL: 27%, KP: 36%, K-only: 37%]. Best overall response differed significantly in the KL (PR: 9.1%, SD: 15.9%, PD: 75%) and KP (PR: 33.3%, SD: 20%, PD: 46.7%) sub-groups (P = 0.005, Fisher’s exact test). PFS was significantly longer in KP compared to KL LUAC (median 12.9 wks vs 8.4 wks, HR = 0.64, 95% CI 0.39-0.95, P = 0.032, log-rank test). ORR in K-only tumors was 21.3% and median PFS 11.4 wks. PD-L1 positivity (≥1%) was more frequent in KP tumors compared to KL (75% vs 22%, P = 0.03, Fisher’s exact test). In syngeneic murine models of KRAS-mutant LUAC, loss of LKB1 promoted resistance to PD-1 inhibitor monotherapy, suggesting a causative role. Conclusions: Mutational inactivation of STK11/LKB1 represents a novel genomic predictor of de novo resistance to immune checkpoint blockade in KRAS-mutant LUAC, whereas TP53 co-mutations are associated with high likelihood of response. Precision immunotherapy will require tailoring to the co-mutation status of individual tumors.

Abstract IA03: Therapeutically-active neoantigen-specific cancer vaccines

Abstract Our past work focused on defining the ability of of the natural immune system to control and shape developing tumors, a process we termed cancer immunoediting. We showed that in its most complex form, cancer immunoediting occurs in three phases: elimination, equilibrium and escape. We subsequently used a combination of exome sequencing and epitope prediction algorithms to show that mutant proteins in highly immunogenic tumor cells derived from methylcholanthrene treated immunodeficient mice represent immunodominant, tumor specific antigens for CD8+ T cells and that immunoselection was a major mechanism underlying the immunoediting process. We next asked whether our approach could be extended into the area of immunotherapy by exploring whether we could identify antigens in progressively growing established tumors that rendered them susceptible to checkpoint blockade immunotherapy. T cell lines generated from anti-PD-1 treated mice that rejected T3 progressor sarcoma cells displayed restriction to H-2Kb but not to H-2Db. We then identified expressed nonsynonymous mutations in T3 cells using exome sequencing and generated a prioritized list of potential H-2Kb binding neoepitopes. This analysis predicted two unequivocal “best candidates”—a mutant form of Laminin α subunit 4 (mLama4) and a mutant glucosyltransferase (mAlg8). When tested in vitro, the two neoepitopes were the only ones among the 62 top predicted H-2Kb binding sequences that stimulated T3 sarcoma specific T cells. These findings were validated by showing that: a) the two mutant neoepitopes could be detected bound to H-2Kb on intact T3 sarcoma cells; b) CTLs expressing TCRs for mLama4 and mAlg8 accumulated in T3 tumors in anti-PD-1-treated, tumor-bearing mice; c) vaccination of naïve WT mice with mutant but not WT forms of Lama4 or Alg8 peptides induced strong CD8+ T cell responses; and d) naïve mice vaccinated either prophylactically or therapeutically with synthetic long peptides containing the mLama4 plus mAlg8 neoepitopes controlled outgrowth of T3 tumors. We have since gone on to explore whether MHC-I restricted neoepitopes were sufficient to drive therapeutic responses to tumors. For this work, we used oncogene driven sarcomas derived from mice expressing a mutant activated form of Kras and lacking p53 (KP sarcomas). KP sarcomas were not immunogenic and their outgrowth could not be controlled in mice treated either with the combination of anti-CTLA-4 plus anti-PD-1 or using a tumor challenge and re-challenge protocol. Exome sequencing of KP sarcomas revealed that they expressed an extremely limited number of nonsynonomous mutations and that none of the mutations were predicted to form epitopes for T cells. Analysis of tumor bearing mice revealed that they did not develop either tumor reactive CD4+ or CD8+ T cells. Enforced expression of strong MHC-I or MHC-II epitopes into KP sarcomas did not increase tumor immunogenicity. In contrast, ectopic expression of combinations of both MHC-I plus MHC-II epitopes in KP tumors rendered the tumor cells highly immunogenic resulting in their rejection. These data thus demonstrate that protective immune responses to tumors requires the presence of both MHC-I and MHC-II restricted epitopes and thus provide additional insights into the requirements for effective personalized cancer vaccines. Citation Format: Robert D. Schreiber. Therapeutically-active neoantigen-specific cancer vaccines [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA03.

Abstract 2403: Molecular mechanisms orchestrating intratumoral heterogeneity in Kras-driven carcinomas

Abstract The considerable variability within tissue microenvironments as well as the multiclonality of cancers leads to intratumoral heterogeneity. This increases the probablility of cellular states that promote resistance to therapy and eventually lead to reconstitution of the tumor by treatment-resistant cancer cells, which in some cases have properties of normal tissue stem cells. Wnt signals are important in the maintenance of stem cells in various epithelial tissues, including in lung development and regeneration. We hypothesized that Wnt signals contribute to tumor heterogeneity in genetically engineered KrasG12D; Tp53Δ/Δ (“KP”) mouse lung adenocarcinomas (LUAD). We observed that a subpopulation of LUAD cells exhibited high Wnt reporter activity and had increased tumor forming ability, which could be suppressed by silencing of Wnt signaling pathway components or by small molecule Wnt inhibitors in vitro and in vivo. KP LUAD cells show hierarchical features with two distinct populations, one with increased Wnt reporter activity and another forming a niche that provides the Wnt signal. Lineage-tracing experiments in the autochthonous KP tumors demonstrated that Wnt responder cells have increased tumor propagation ability in vivo. Strikingly, selective ablation of the Wnt responder cells resulted in tumor stasis. CRISPR-based targeting or small molecules targeting Wnt signaling reduced tumor growth and prolonged survival in the autochthonous KP mouse lung cancer model. Interestingly, we found that a similar Wnt-driven mechanism operates to establish an intratumoral hierarchy also in other Kras-driven carcinomas. These results indicate that maintenance of heterogeneity within tumors may be advantageous for the tumor cell population collectively, which may be utilized as a therapeutic target to homogenize cancer cell phenotypes within tumors. Citation Format: Roxana Azimi. Molecular mechanisms orchestrating intratumoral heterogeneity in Kras-driven carcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2403.

Combination wt-p53 and MicroRNA-125b Transfection in a Genetically Engineered Lung Cancer Model Using Dual CD44/EGFR-targeting Nanoparticles

Mutations in KRAS and p53 signaling pathways contribute to loss of responsiveness to current therapies and a decreased survival in lung cancer. In this study, we have investigated the delivery and transfection of wild-type (wt-) p53 and microRNA-125b (miR-125b) expressing plasmid DNA, in SK-LU-1 human lung adenocarcinoma cells as well as in Kras(G12D)/p53(fl/fl) (KP) genetically engineered mouse model of lung cancer. Systemic plasmid DNA delivery with dual CD44/EGFR-targeted hyaluronic acid (HA)-based nanoparticles (NPs) resulted in a 2- to 20-fold increase in wt-p53 and miR-125b gene expression in SK-LU-1 cells. This resulted in enhanced apoptotic activity as seen with increased APAF-1 and caspase-3 gene expression. Similarly, in vivo evaluations in KP mouse model indicated successful CD44/EGFR-targeted delivery. Tumor growth inhibition and apoptotic induction were also observed with (wt-p53+miR125b) combination therapy in KP tumor model. Lastly, J774.A1 murine macrophages co-cultured with transfected SK-LU-1 cells showed a 14- to 35-fold increase in the iNOS-Arg-1 ratio, supportive of previous results demonstrating a role of miR-125b in macrophage repolarization. Overall, these results show tremendous promise of wt-p53 and miR-125b gene therapy using dual CD44/EGFR-targeting HA NP vector for effective treatment of lung cancer.

Abstract IA05: Personalizing cancer immunotherapy

Abstract Our past work focused on defining the ability of the natural immune system to control and shape developing tumors, a process we termed Cancer Immunoediting. We showed that in its most complex form, Cancer Immunoediting occurs in three phases: Elimination, Equilibrium and Escape. We subsequently used a combination of exome sequencing and epitope prediction algorithms to show that mutant proteins in highly immunogenic tumor cells derived from methylcholanthrene treated immunodeficient mice represent immunodominant, tumor specific antigens for CD8+ T cells and that immunoselection was a major mechanism underlying the immunoediting process. We next asked whether our approach could be extended into the area of immunotherapy by exploring whether we could identify antigens in progressively growing established tumors that rendered them susceptible to checkpoint blockade immunotherapy. T cell lines generated from anti-PD-1 treated mice that rejected T3 progressor sarcoma cells displayed restriction to H-2Kb but not to H-2Db. We then identified expressed nonsynonymous mutations in T3 cells using exome sequencing and generated a prioritized list of potential H-2Kb binding neoepitopes. This analysis predicted two unequivocal “best candidates” a mutant form of Laminin α subunit 4 (mLama4) and a mutant glucosyltransferase (mAlg8). When tested in vitro, the two neoepitopes were the only ones among the 62 top predicted H-2Kb binding sequences that stimulated T3 sarcoma specific T cells. These findings were validated by showing that: (i) the two mutant neoepitopes could be detected bound to H-2Kb on intact T3 sarcoma cells; (ii) CTLs expressing TCRs for mLama4 and mAlg8 accumulated in T3 tumors in anti-PD-1-treated, tumor-bearing mice; (iii) vaccination of naïve WT mice with mutant but not WT forms of Lama4 or Alg8 peptides induced strong CD8+ T cell responses; and (iv) naïve mice vaccinated either prophylactically or therapeutically with synthetic long peptides containing the mLama4 plus mAlg8 neoepitopes controlled outgrowth of T3 tumors. We have since gone on to explore whether MHC-I restricted neoepitopes were sufficient to drive therapeutic responses to tumors. For this work, we used oncogene driven sarcomas derived from mice expressing a mutant activated form of Kras and lacking p53 (KP sarcomas). KP sarcomas were not immunogenic and their outgrowth could not be controlled in mice treated either with the combination of α-CTLA-4 plus αPD-1 or using a tumor challenge and re-challenge protocol. Exome sequencing of KP sarcomas revealed that they expressed an extremely limited number of nonsynonomous mutations and that none of the mutations were predicted to form epitopes for T cells. Analysis of tumor bearing mice revealed that they did not develop either tumor reactive CD4+ or CD8+ T cells. Enforced expression of strong MHC-I or MHC-II epitopes into KP sarcomas did not increase tumor immunogenicity. In contrast, ectopic expression of combinations of both MHC-I plus MHC-II epitopes in KP tumors rendered the tumor cells highly immunogenic resulting in their rejection. These data thus demonstrate that protective immune responses to tumors requires the presence of both MHC-I and MHC-II restricted epitopes and thus provide additional insights into the requirements for effective personalized cancer vaccines. Citation Format: Robert D. Schreiber. Personalizing cancer immunotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr IA05.

Abstract B120: Characterization and combination immunotherapy treatment of an inducible autochthonous murine lung cancer model expressing human carcinoembryonic antigen (CEA) as a tumor-associated self-antigen

Abstract Previous work from our lab has demonstrated that a combination of anti-tumor antibody and an IL-2 fusion protein that exhibits extended serum half-life elicits an immune response that can effectively control a wide variety of tumor models. We have since then combined this therapeutic regimen with a vaccine exhibiting efficient lymph node trafficking that can generate an impressive population of tumor-antigen specific CD8+ T-cells but by itself does not provide good anti-tumor efficacy. The efficacy of this combination immunotherapy is further boosted by immune checkpoint blockade, leading to a robust four-component therapy: 1) anti-tumor antigen antibody; 2) IL-2 fusion protein; 3) anti-tumor antigen vaccine; 4) anti-PD-1 antibody. Although this four-pronged approach is demonstrably effective in the syngeneic subcutaneous melanoma model B16F10, we wish to test its efficacy in a more physiological model. To this end, we have turned to a model developed by the Jacks Lab, known as the KP model: an inducible lung tumor model where lentivirus-driven integration and expression of Cre is able to activate oncogenic Kras and completely remove p53 function. Because our therapeutic regimen requires a targetable tumor-associated antigen with respect to both the antibody and vaccine, we chose to induce expression of human carcinoembryonic antigen (CEA) in these tumors, as CEA has a well-studied structure and biology, and frequently expresses aberrantly in many forms of human adenocarcinomas. Additionally, our lab has previously engineered an antibody targeting CEA possessing picomolar affinity. Finally, to remove any endogenous immunological response against human CEA as a foreign antigen in our mouse system, we have crossed the KP model with a mouse model transgenic for human CEA, which in the literature has been described to have identical spatiotemporal expression of CEA as found in humans and should allow for central tolerance of this antigen. In the course of this work, we have successfully introduced human CEA into our lentivirus constructs and shown tumorigenesis by these constructs in the KP model coincides with expression of tumor-associated CEA, as detected by qPCR. On the therapeutic side, we have tailored the vaccine to successfully drive an anti-CEA CD8+ T-cell response. Performing preliminary therapeutic experiments in a transplant model of the KP tumor with our four-component therapy, we saw tumor control compared to untreated tumors. Upon interrogating the CD8+ T-cell response against CEA, we found 1-15% of CD8+ T-cells in the blood respond to CEA stimulation by intracellular cytokine staining. With regards to the lung tumor model, in the course of establishing the system we have also observed that the growth kinetics of tumors expressing CEA lags behind those tumors without CEA, even in the transgenic background. Preliminary immunophenotyping work by flow cytometry suggests that tumors with CEA seem to have a reduced myeloid-derived suppressor cell (MDSC) population and a higher CD8a+ dendritic cell (DC) population compared to tumors without CEA, suggesting that the former may have a less immunosuppressive tumor microenvironment that is better able to prime an anti-tumor CD8+ T-cell response. We will be planning to conduct therapeutic trials in the more physiological lung tumor KP model in the near future, as well as investigate the differences in the immune response with tumors expressing or lacking CEA. Citation Format: Eric F. Zhu, Kavya Rakhra, Naveen Mehta, Kelly D. Moynihan, Cary F. Opel, Darrell J. Irvine, K. Dane Wittrup. Characterization and combination immunotherapy treatment of an inducible autochthonous murine lung cancer model expressing human carcinoembryonic antigen (CEA) as a tumor-associated self-antigen. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B120.

Abstract 968: Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma (LUAC) with distinct biology and therapeutic vulnerabilities

Abstract Introduction The development of more effective treatment strategies for LUAC bearing activating mutations in KRAS is hampered by the biological heterogeneity of KRAS-mutant tumors. The molecular underpinnings that drive this process are poorly characterized. Here, we implemented an integrated approach to the discovery of biologically distinct subsets of KRAS-mutant LUAC and explored their molecular vulnerabilities. Methods Our datasets consisted of 68 KRAS-mutant tumors from TCGA, 88 additional chemo-naive KRAS-mutant LUACs (PROSPECT and Chitale datasets) and 36 platinum-refractory LUACs from the BATTLE-2 clinical trial. Non-negative matrix factorization (NMF) consensus clustering was applied to RNASeq data as previously described. Signature enrichment was assessed using Gene Set Enrichment Analysis (GSEA). Results NMF consensus clustering identified three robust subsets of KRAS-mutant LUAC that were reproducible across diverse clinical datasets of early-stage, chemotherapy-naive and metastatic, chemo-refractory tumors. Distinct KRAS-mutant alleles were not differentially represented in the three subgroups (P = 0.3). In contrast, the subgroups were dominated, respectively, by co-occurring genetic events in STK11/LKB1 (termed the KL subgroup) (P = 1.03e−05), TP53 (KP) (P = 3.8e-06) and low expression of TTF1 coupled with frequent CDKN2A/B inactivation (KC) (P = 0.004 and P = 0.002). Distinct patterns of intracellular signaling were detected in the three subsets. KL tumors showed evidence of LKB1-AMPK pathway inactivation and adaptation to energetic, proteotoxic and oxidative stress, the latter exemplified by near ubiquitous inactivation of KEAP1 and up-regulation of a NRF2-driven antioxidant signature. KP tumors carried a higher somatic mutation load and were characterized by prominent inflammation and up-regulation of several immune checkpoint effector molecules, including PD-L1. KC tumors frequently displayed a GI-like differentiation program, suppression of MTORC1 signaling and elevated wild-type p53 transcriptional output. Using a large panel of KRAS-mutant NSCLC cell lines we detected co-mutation-dependent patterns of drug sensitivity. Specifically, KL cell lines showed enhanced sensitivity to several structurally distinct HSP90 inhibitors. These results were confirmed in panels of isogenic cell lines. Mechanistically, treatment with ganetespib resulted in concurrent degradation of several molecules with established role in supporting the fitness of LKB1-deficient cells. Conclusions Our work identifies three major subsets of KRAS-mutant LUAC - dominated by co-occurring genetic events - with distinct biology and therapeutic vulnerabilities. Citation Format: Ferdinandos Skoulidis, Lauren Byers, Lixia Diao, Vassiliki Papadimitrakopoulou, Pan Tong, Julie Izzo, Carmen Behrens, Humam Kadara, Edwin R. Parra, Jaime Rodriguez-Canales, Jianjun Zhang, Uma Giri, Jayanthi Gudikote, Maria Angelica Cortez, Chao Yang, You Hong Fan, Michael Peyton, Luc Girard, Kevin R. Coombes, Carlo Toniatti, Timothy P. Heffernan, Murim Choi, Garrett M. Frampton, Vincent Miller, John N. Weinstein, Roy S. Herbst, Kwok-Kin Wong, Jianhua Zhang, Padmanee Sharma, Gordon M. Mills, Waun Ki Hong, John D. Minna, James P. Allison, Andrew Futreal, Jing Wang, Ignacio Wistuba, John V. Heymach. Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma (LUAC) with distinct biology and therapeutic vulnerabilities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 968. doi:10.1158/1538-7445.AM2015-968

Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma with distinct biology, immune profiles, and therapeutic vulnerabilities.

The molecular underpinnings that drive the heterogeneity of KRAS-mutant lung adenocarcinoma are poorly characterized. We performed an integrative analysis of genomic, transcriptomic, and proteomic data from early-stage and chemorefractory lung adenocarcinoma and identified three robust subsets of KRAS-mutant lung adenocarcinoma dominated, respectively, by co-occurring genetic events in STK11/LKB1 (the KL subgroup), TP53 (KP), and CDKN2A/B inactivation coupled with low expression of the NKX2-1 (TTF1) transcription factor (KC). We further revealed biologically and therapeutically relevant differences between the subgroups. KC tumors frequently exhibited mucinous histology and suppressed mTORC1 signaling. KL tumors had high rates of KEAP1 mutational inactivation and expressed lower levels of immune markers, including PD-L1. KP tumors demonstrated higher levels of somatic mutations, inflammatory markers, immune checkpoint effector molecules, and improved relapse-free survival. Differences in drug sensitivity patterns were also observed; notably, KL cells showed increased vulnerability to HSP90-inhibitor therapy. This work provides evidence that co-occurring genomic alterations identify subgroups of KRAS-mutant lung adenocarcinoma with distinct biology and therapeutic vulnerabilities. Co-occurring genetic alterations in STK11/LKB1, TP53, and CDKN2A/B-the latter coupled with low TTF1 expression-define three major subgroups of KRAS-mutant lung adenocarcinoma with distinct biology, patterns of immune-system engagement, and therapeutic vulnerabilities.

Abstract 100: Establishment and characterization of allografts derived from a genetically engineered mouse model of non-small cell lung cancer (NSCLC)

Abstract Introduction: Genetically engineered mouse models (GEMM) represent an attractive system for preclinical research since GEMM tumors develop in the presence of a competent immune system, thereby closely resembling the tumor microenvironment in patients. One example is the KP model (DuPage et al., 2009), which reflects the tumorigenesis of NSCLC in humans. KP tumors carry Kras and Trp53 mutations, comparable to subtypes of human NSCLC. Typical drawbacks of GEMM tumors include differences in their genetic make-up to human tumors and the very slow and heterogeneous tumor development, making the use of these models challenging for routine in vivo efficacy studies. The latter can be improved by grafting primary tumors on a genetically dissimilar member of the same strain, so-called allografting. Thus, the advantages of xenograft and GEMM models can be combined. Here, we described the establishment and characterization of allografts from the KP GEMM. Material & Methods: KP tumors were excised from the lungs of KP mice and implanted subcutaneously (s.c.) into C57BL/6N female mice. When tumor growth was detected, passaging steps from animal to animal were performed. Furthermore, two cell lines derived from KP tumors, KP1 and KP4 (provided by Müller et al.), were injected s.c., the developing tumors excised and directly re-implanted as described above. The passages were analyzed molecularly, histologically, and via IHC (e.g. Ki67 proliferation marker) , and compared to the original in-situ tumors. A therapeutic study with Erlotinib and BEZ235 was performed on KP1 allografted mice. Results: All investigated allografts showed similar histological patterns. Ki67 scores reflected the average time from implantation to passaging. The average passage times ranged from 19±3 days (KP4 cell line) to 26±6 days (KP1 cell line). Allografts from the original KP model showed a time to passaging of 23±5 days. The mRNA expression of EpCAM (CD326) was on average 10-fold higher in KP and KP1 allografted tumors compared to normal C57BL/6 lung tissue, which could be confirmed by flow cytometry and cell-binding assay, determining >99% EPCAM positive cells in KP1 tumors. All allografts carried the Kras mutation and loss of function point mutations in Trp53. The therapy study revealed no effect of Erlotinib on tumor growth, but a significant (p=0.0003) tumor load reduction in mice treated with BEZ235 (519 ± 244 mm³, n=5) compared to the control group (1678 ± 275 mm³, n=4) on day 28, respectively. Conclusion: The generation of s.c. transplantable allografts from the KP GEMM was successfully conducted. The allograft models enable the use of immunocompetent mice in a feasible time frame and still resemble key aspects of human disease. Further studies will elucidate the interaction of the allografts with the host and compare sensitivity characteristics between GEMM and allograft of the same model. Citation Format: Damaris Kukuk, Philipp Müller, Kerstin Klingner, Anne-Lise Peille, Alfred Zipelius, Julia B. Schüler. Establishment and characterization of allografts derived from a genetically engineered mouse model of non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 100. doi:10.1158/1538-7445.AM2014-100

A Soluble BAFF Antagonist, BR3-Fc, Decreases Peripheral Blood B Cells and Lymphoid Tissue Marginal Zone and Follicular B Cells in Cynomolgus Monkeys

BAFF (also known as BLyS), a member of the tumor necrosis factor superfamily, plays a critical role in the maturation and development of B cells. BAFF has three receptors on B cells, the most crucial of which is BR3. In this study, we demonstrate the biological outcome of BAFF blockade in cynomolgus monkeys using a soluble fusion protein consisting of human BR3 and human IgG1 Fc. In vitro, BR3-Fc blocked BAFF-mediated survival and proliferation of cynomolgus monkey B cells. Weekly treatment of cynomolgus monkeys with BR3-Fc for 13 to 18 weeks resulted in significant B-cell reduction in the peripheral blood and in lymphoid organs. CD21(high) B cells in lymphoid tissues, a subset analogous to human marginal zone B cells, expressed nearly twofold higher BR3 levels than did CD21(med) B cells. Lymphoid tissue flow cytometric analysis showed that BR3-Fc reduced this CD21(high) B-cell subset to a greater extent than it reduced CD21(med) B cells. Dual-label immunohistochemistry and morphometric image analysis supported these results by demonstrating that BR3-Fc reduced a significant proportion of the B cells within the splenic inner and outer marginal zones. These findings should prove very useful in guiding the desired therapeutic use of BR3-Fc for autoimmune diseases in the clinic.