Rational Combination Strategies Research Articles

Ibrutinib and venetoclax target distinct subpopulations of CLL cells: implication for residual disease eradication

Ibrutinib inhibits Bruton tyrosine kinase while venetoclax is a specific inhibitor of the anti-apoptotic protein BCL2. Both drugs are highly effective as monotherapy against chronic lymphocytic leukemia (CLL), and clinical trials using the combination therapy have produced remarkable results in terms of rate of complete remission and frequency of undetectable minimal residual disease. However, the laboratory rationale behind the success of the drug combination is still lacking. A better understanding of how these two drugs synergize would eventually help develop other rational combination strategies. Using an ex vivo model that promotes CLL proliferation, we show that modeled ibrutinib proliferative responses, but not viability responses, correlate well with patients’ actual clinical responses. Importantly, we demonstrate for the first time that ibrutinib and venetoclax act on distinct CLL subpopulations that have different proliferative capacities. While the dividing subpopulation of CLL responds to ibrutinib, the resting subpopulation preferentially responds to venetoclax. The combination of these targeted therapies effectively reduced both the resting and dividing subpopulations in most cases. Our laboratory findings help explain several clinical observations and contribute to the understanding of tumor dynamics. Additionally, our proliferation model may be used to identify novel drug combinations with the potential of eradicating residual disease.

Overcoming TKI resistance in fusion-driven NSCLC: new generation inhibitors and rationale for combination strategies.

During the last several years, multiple gene rearrangements with oncogenic potential have been described in NSCLC, identifying specific clinic-pathological subgroups of patients that benefit from a targeted therapeutic approach, including anaplastic lymphoma kinase (ALK), c-ros protooncogene 1 (ROS1) and, more recently, REarranged during Transfection (RET) and neurotrophic tyrosine receptor kinases (NTRK) genes. Despite initial impressive antitumor activity, the use of targeted therapies in oncogene-addicted NSCLC subgroups is invariably associated with the development of acquired resistance through multiple mechanisms that can include both on-target and off-target mechanisms. However, the process of acquired resistance is a rapidly evolving clinical scenario that constantly evolves under the selective pressure of tyrosine kinase inhibitors. The development of increasingly higher selective and potent inhibitors, traditionally used to overcome resistance to first generation inhibitors, is associated with the development of novel mechanisms of resistance that encompass complex resistance mutations, highly recalcitrant to available TKIs, and bypass track mechanisms. Herein, we provide a comprehensive overview on the therapeutic strategies for overcoming acquired resistance to tyrosine kinase inhibitors (TKIs) targeting the most well-established oncogenic gene fusions in advanced NSCLC, including ALK, ROS1, RET, and NTRK rearrangements.

EXTH-75. IN VITRO AND IN VIVO EFFICACY OF COMBINATORIAL INHIBITION OF LSD1 AND HDACS IN PATIENT DERIVED GLIOBLASTOMA STEM CELL MODELS

Abstract Inhibitors of histone deacetylases (HDACi) have been tested in glioblastoma (GBM), however, single agent clinical efficacy has not been proven, prompting study of combinatorial approaches. A rational HDACi combination strategy is with inhibition of LSD1, lysine specific demethylase 1, a histone demethylase known to exist in complex with HDAC1/2. We previously showed in vivo efficacy of combining a brain permeant LSD1 inhibitor, tranylcypromine (TCP) together with vorinostat. More selective inhibitors of LSD1 have been developed and were tested in the current study together with the HDACi, vorinostat or panobinostat in a panel of patient derived glioblastoma stem cell (GSC) lines that have been characterized as radio-resistant or radiosensitive. To test in vitro sensitivity, dose response experiments were conducted in nine GSC cell lines using three LSD1 inhibitors or two HDACi as single agents or in combination. Two non-tumor lines were also tested to assess selective cytotoxicity for brain tumor lines. Cell viability and clonogenicity assays were performed. Several radio-resistant GSC lines showed sensitivity to single agent LSD1 inhibitors, whereas some radio-sensitive GSCs did not, indicating distinct molecular mechanisms of response. Intracranial orthotopic xenograft models were used to test in vivo efficacy using the radioresistant GSC20 line. Interestingly, LSD1 inhibition alone promoted tumor burden reduction and better survival rates than combination treatment. RNA-Seq guided biomarkers of sensitivity to the LSD1/HDAC inhibitor combination were assessed in vitro and in vivo. Both in vivo and in vitro data show that GSC20 treated with GSK LSD1 inhibitor had higher levels of HKDC1 gene with in vivo data also showing elevated levels of FTH1. In vivo mouse data for vorinostat and combination treatment groups showed that only the FTH1 gene level was elevated, providing a potential explanation for low in vivo efficacy. Collectively, our data suggests that LSD1 inhibition represents a viable strategy in GBM.

Abstract 2788: Characterization of a panel of orthotopic syngeneic models using bioluminescent imaging for the evaluation of immuno oncology therapeutics

Abstract Background: The rapid development of immuno-oncology (I/O) therapies for various cancer types has transformed the paradigm of cancer treatment from targeting the tumor to targeting the immune system, and now to combination strategies. Numerous Phase I-III trials with immunotherapeutics and combinations are being conducted; however, a lot of these are failing. More preclinical models are required to help investigate efficacy, uncover mechanisms of action, and to design more rational combination strategies. The most widely used models for immunotherapy evaluation are models with functional murine immunity such as syngeneic allograft models and genetically engineered mouse models (GEMMs). Syngeneic models are used in nearly all I/O therapy programs as these are well-characterized and straightforward to set-up as subcutaneous models. However, one of the major disadvantages of subcutaneous models is the lack of a clinically relevant tumor microenvironment (TME) including stromal cells, immune cells, extracellular matrix etc. The dynamic interplay of these cells drive alterations in cellular functions and impact study outcomes. Using bioluminescent imaging (BLI), we have developed and characterized a panel of orthotopic models where tumor cells are inoculated in a relevant organ-specific location to recapitulate the immune and stromal component interactions with the tumor, which can also facilitate metastatic spread. These models can be used to evaluate various therapies in both the orthotopic and metastatic settings. Method: A panel of syngeneic cell lines were labeled with bioluminescence by transducing the cell lines with a lentiviral vector carrying the firefly luciferase gene. This panel represents a diverse range of cancer types including liver (Hepa 1-6 and HT22), breast (4T1), colon (CT26.WT), brain (GL261), prostate (RM-1), and pancreas (Pan02). These bioluminescent cells were orthotopically implanted into immune competent mice. In-life tumor growth and metastasis were assessed using the IVIS® Spectrum In Vivo Imaging System. Tumor-infiltrating lymphocytes were assessed by flow cytometry (BD LSRFortessa™) and multiplex IHC. Results: Orthotopic implantation of syngeneic models was successfully demonstrated for all models as confirmed by in-life imaging and ex vivo imaging at termination. Baseline response towards various checkpoint inhibitors and chemotherapies were established. For the Hepa 1-6 liver model, response to an anti-PD-1 antibody was significant in both the subcutaneous and orthotopic setting. In contrast, the H22 liver model responded to both sorafenib and anti-mCTLA-4 antibody in the subcutaneous model but not in the orthotopic setting. In addition, anti-mCTLA-4 treatment led to more significant inhibition of pancreatic Pan02 tumor growth in the orthotopic model in comparison with its corresponding subcutaneous setting. Conclusion: Bioluminescent syngeneic models in the orthotopic setting provide a valuable tool for testing immunotherapies, with a more clinically relevant TME in comparison to the corresponding subcutaneous model. The establishment of a range of models covering diverse cancer types allows for a thorough interrogation of immunotherapeutic potential. Citation Format: Diandong Jiang, Bryan Miller, Yanrui Song, Rajendra Kumari, Annie An, Jie Cai, Davy Xuesong Ouyang, Henry Qixiang Li, Yinfei Yin. Characterization of a panel of orthotopic syngeneic models using bioluminescent imaging for the evaluation of immuno oncology therapeutics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2788.

Abstract 1027: Inhibition of c-MET upregulates PD-L1 related immune escape in lung adenocarcinoma

Abstract Non-small cell lung cancer (NSCLC) patients with c-MET dysregulation may benefit from c-MET inhibitors therapy as inhibition of c-MET activity has emerged as a therapeutic approach against this disease. Although several c-MET inhibitors have been evaluated in phase III clinical trials in lung cancer, their benefits so far have been modest. Thus, furthering our understanding of the mechanisms contributing to the lack of success of c-MET inhibitors in clinical trials is essential toward the development of rational and effective combination strategies. Here we show that exposure of NCSLC cell lines to c-MET inhibitor tivantinib increases their expression of PD-L1, which in turn causes cells to become more resistant to T-cell killing. Tivantinib upregulates PD-L1 in NSCLC cells in dose and time-dependent manner. Mechanistically, inhibition of c-MET suppresses p-GSK3-beta, leading to the stabilization of PD-L1. Collectively, our findings suggest a potential crosstalk between c-Met inhibition and immune escape and provide a rationale for the combination therapy of c-Met inhibitors and immune checkpoint blockade in NSCLC. Citation Format: Xian Sun, Chia-Wei Li, Wei-Jan Wang, Mien-Chie Hung. Inhibition of c-MET upregulates PD-L1 related immune escape in lung adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1027.

Abstract 2837: Dual inhibition of SHP2 and CDK4/6 leads to immunological memory and immune-mediated anti-tumor activity in a mouse syngeneic model of breast cancer

Abstract Allosteric inhibition of the phosphatase SHP2, a key signaling node in the RAS pathway, is a novel, investigational therapeutic strategy for patients bearing tumors with specific oncogenic mutations in this pathway. SHP2 also participates in signal transduction downstream of regulatory immunoreceptors and we have previously shown that inhibition of SHP2 drives anti-tumor immunity through modulation of both innate and adaptive mechanisms in preclinical models (Quintana E. et al, A5019, AACR2019). Rational combination strategies offer an opportunity to optimize the therapeutic potential of SHP2 inhibitors. For example, we have demonstrated that SHP2 inhibition has combinatorial anti-tumor effects with a CDK4/6 inhibitor in several RAS-driven human cancer cell-line derived xenograft models (Lee G. et al, A1322, AACR2019); however, these models use immunocompromised mice, making it impossible to assess an effect of the combination on anti-tumor immunity. To evaluate the impact of dual SHP2 and CDK4/6 inhibition, we tested this combination in immunocompetent EMT6 tumor-bearing mice. EMT6 is a RAS-driven model of triple-negative breast cancer, which is partially sensitive to SHP2 or CDK4/6 inhibition via cell intrinsic mechanisms. While monotherapy treatment with either RMC-4550, an allosteric inhibitor of SHP2, or abemaciclib, a CDK4/6 inhibitor, resulted in modest tumor growth inhibition, combination treatment led to complete tumor regressions in 40% (4 of 10) of the mice. Furthermore, 30% (3 of 10) of the mice remained tumor-free upon cessation of treatment and, when re-challenged with EMT6 cells tumors failed to grow demonstrating that this dual SHP2-CDK4/6 inhibition results in formation of immunological memory. Combination treatment was well tolerated in mice. Recently, CDK4/6 inhibitors have been shown to augment anti-tumor immunity via a number of mechanisms, including increases in antigen presentation by the tumor and enhancement of T cell activation. Analysis of the immune landscape in the tumor microenvironment nine days after treatment revealed that RMC-4550 increased the frequency of CD8+T cells and depleted F4/80+ macrophages in EMT6 tumors, and the combination with abemaciclib amplified these changes. Our results suggest that dual SHP2 and CDK4/6 targeting is a promising investigational strategy against tumors driven by mutations in the RAS signaling pathway that combines direct inhibition of cancer cell growth with immunomodulation to potentially achieve robust and durable anti-tumor responses. Citation Format: Nataliya Tovbis Shifrin, Grace J. Lee, Neil Omaque, Tram Nguyen, Amira Belwafa, Edward G. Lorenzana, Robert J. Nichols, Jacqueline A. Smith, Mallika Singh, Elsa Quintana. Dual inhibition of SHP2 and CDK4/6 leads to immunological memory and immune-mediated anti-tumor activity in a mouse syngeneic model of breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2837.

Metformin Synergistically Enhanced the Antitumor Activity of Celecoxib in Human Non-Small Cell Lung Cancer Cells.

Celecoxib has potential as an effective antineoplastic agent, but it may exhibit side effects. Given the glucose-addicted properties of tumor cells, metformin is recognized for its inhibitory effect on oxidative phosphorylation. In the present study, we aimed to combine low dose of celecoxib with metformin to alleviate the side effects of nonsteroidal anti-inflammatory drugs (NSAIDs) and overcome potential drug resistance. We found that celecoxib combined with metformin obviously suppressed cell migration and proliferation and induced cell apoptosis. Most importantly, in vivo experiments revealed the superior antitumor efficacy of combination treatment with a low dosage of celecoxib (25 mg/kg/day) without apparent toxicity. Further study of the underlying mechanism revealed that the two drugs in combination caused ROS aggregation in NSCLC cells, leading to DNA double-strand breaks and increased expression of the tumor suppressor factor p53. Elevated p53 subsequently caused cell cycle arrest and cell proliferation inhibition. The presence of metformin also sensitized NSCLC cells to celecoxib-induced apoptosis by activating caspase-9, -8, -3, and -7, upregulating the pro-apoptotic proteins Bad and Bax, and downregulating the antiapoptotic proteins Bcl-xl and Bcl-2. Moreover, the superior anticancer effect of combined therapy was also due to suppression of Raf-MEK-ERK cascades and PI3K-AKT signaling, which is conducive to overcoming drug resistance. In addition, either celecoxib alone or in combination with metformin suppressed NSCLC cell migration and invasion by inhibiting FAK, N-cadherin, and matrix metalloproteinase-9 activities. Together, our study provided a rational combination strategy with a low dosage of celecoxib and metformin for preclinical cancer application.

Abstract IA19: Immunotherapy in ovarian cancer: Challenges and novel opportunities

Abstract IA19: Immunotherapy in ovarian cancer: Challenges and novel opportunities

Characterization of 1,387 NSCLCs with MET exon 14 (METex14) skipping alterations (SA) and potential acquired resistance (AR) mechanisms.

9511 Background: METex14 SA are oncogenic drivers in NSCLC. Due to the numerous sites around ex14 that bind the spliceosome complex, many variations can result in deleterious alterations (alts). We present a comprehensive overview of these ex14 SA across 1,387 NSCLCs and characterized potential AR mechanisms. Methods: Hybrid-capture based comprehensive genomic profiling (CGP) was performed on samples from 60,495 NSCLC patients (pts). A scoring system was applied leveraging our large database of samples with METex14 SA to optimize accurate reporting of these variants. Paired samples were collected ≥ 60 days apart (median 462). Results: 1,393 METex14 SA were identified in samples (1,278 tissue, 109 circulating tumor DNA (ctDNA)) from 1,387 NSCLC pts (2.3%) spanning multiple functional sites: donor (42%), acceptor (4.7%), poly-pyrimidine tract (15%), acceptor and polypyrimidine tract (13%), D1010 (23%), Y1003 (2.1%), and whole exon deletions (0.3%). 6 samples (5 tissue, 1 ctDNA) harbored 2 METex14 SA, each including a mutation (mut) at the donor or acceptor site. MDM2 and CDK4 amplifications (amps) co-occurred in 32% and 19% of METex14 samples, respectively, but were more common with polypyrimidine tract (37% and 23%) vs donor site (32%, p = 0.07 and 18%, p = 0.07) alts. MET co-amp was present in 12% of cases and frequency did not significantly differ by functional site. 66 (4.8%) cases (57 tissue, 9 ctDNA) had known NSCLC co-drivers, including KRAS (68%) and EGFR (14%) mut, a subset of which may represent AR. Paired samples with a METex14 SA in the 1st sample were available for 36 pts. The METex14 SA was detected in the 2nd sample for 32 pts, excluding 3 with low ctDNA. 22/36 (61%) had reportable acquired alts detected including 9 with ≥1 acquired MET muts [D1228X (4), Y1230X (3), Y1003F (1), D1228A/E/H + L1195V (1)] and 3 with acquired MET amp. Other acquired alts included ERBB2 amp and mut (1 each), EGFR ex19ins (1), KRAS amp (1), PIK3CA mut (1), AKT2 amp (1) and others with unknown functional significance. Potential AR alts were present with primary METex14 SA spanning all functional sites. Conclusions: In a dataset of > 60,000 advanced NSCLCs, METex14 SA were present in 2.3% of cases, and represented 6 major subtypes. Among paired cases, potential AR mechanisms included secondary MET alts (33%), and acquired alts in EGFR, ERBB2, KRAS, and PI3K pathways. Acquired alts were independent of the type of METex14 SA. Characterizing common co-occuring may be critical for predicting responses to MET inhibitors and informing rational combination strategies.

Inhibition of protein phosphatase-2A with LB-100 enhances antitumor immunity against glioblastoma.

Glioblastoma (GBM) carries a dismal prognosis despite standard multimodal treatment with surgery, chemotherapy and radiation. Immune checkpoint inhibitors, such as PD1 blockade, for treatment of GBM failed to show clinical benefit. Rational combination strategies to overcome resistance of GBM to checkpoint monotherapy are needed to extend the promise of immunotherapy to GBM management. Emerging evidence suggests that protein phosphatase 2A (PP2A) plays a critical role in the signal transduction pathways of both adaptive and innate immune cells and that inhibition of PP2A could enhance cancer immunity. We investigated the use of a PP2A inhibitor, LB-100, to enhance antitumor efficacy of PD1 blockade in a syngeneic glioma model. C57BL/6 mice were implanted with murine glioma cell line GL261-luc or GL261-WT and randomized into 4 treatment arms: (i) control, (ii) LB-100, (iii) PD1 blockade and (iv) combination. Survival was assessed and detailed profiling of tumor infiltrating leukocytes was performed. Dual PP2A and PD1 blockade significantly improved survival compared with monotherapy alone. Combination therapy resulted in complete regression of tumors in about 25% of mice. This effect was dependent on CD4 and CD8 T cells and cured mice established antigen-specific secondary protective immunity. Analysis of tumor lymphocytes demonstrated enhanced CD8 infiltration and effector function. This is the first preclinical investigation of the effect of combining PP2A inhibition with PD1 blockade for GBM. This novel combination provided effective tumor immunotherapy and long-term survival in our animal GBM model.

A35 Dendritic Cell in Situ Vaccination Potentiates Anti-PD-1 Efficacy and Induces Immunoediting in a Murine Model of NSCLC

Studies reveal that responses to checkpoint blockade in non-small cell lung cancer (NSCLC) are associated with high tumor mutational burden (TMB), preexisting CD8+ T-cell infiltration, and high baseline PD-L1 expression within the tumor microenvironment (TME). In contrast, co-occurring KRAS/LKB1 mutation is associated with primary resistance to PD-1 blockade and decreased overall survival. In preclinical studies as well as a phase I clinical trial, we have discovered that intratumoral (IT) vaccination with gene-modified dendritic cells expressing CCL21 (CCL21-DC) promotes tumor effector T-lymphocyte infiltration, PD-L1 upregulation, and systemic tumor-specific immune responses. We hypothesized that in situ vaccination with CCL21-DC could restore tumor antigen presentation and promote T-cell priming and activation, thereby sensitizing nonresponsive NSCLC tumors to checkpoint blockade. Although genetically engineered murine models (GEMMs) of NSCLC bear driver mutations of the disease, recent studies reveal that these GEMMs possess low mutational burden. We established novel GEMMs of NSCLC [KrasG12D (K), KrasG12DP53-/- (KP), KrasG12DP53+/-Lkb1-/- (KPL)] bearing common driver mutations and varying mutational loads by in vitro exposure of tumor cell lines to the carcinogen N-methyl-N-nitrosourea (MNU). Our preclinical KPL model with high TMB recapitulates the immunologic phenotype of human disease, and contains a predominance of myeloid-derived suppressor cells (MDSC), low tumor-infiltrating lymphocytes (TILs), and low PD-L1 expression within the TME. As anticipated, the KPL tumors are resistant to anti-PD-1 therapy, even with increased mutational load. We evaluated IT CCL21-DC combined with anti-PD-1 therapy in immunocompetent mice bearing KPL tumors with high TMB, and observed that IT CCL21-DC vaccination induces infiltration of autologous T lymphocytes and conventional type I DCs (cDC1s) into the TME and sensitizes the tumors to anti-PD-1 therapy. Combination therapy also reprogrammed the myeloid compartment, resulting in a significant reduction of MDSCs and a concurrent increase in CD11b+Ly6GhiLy6Clo monocyte/myeloid population. Whole-exome sequencing (WES) of tumors revealed immunoediting and selective depletion of tumor subclones post IT CCL21-DC and anti-PD1 combination therapy. Future studies will evaluate the evolution of the T-cell receptor (TCR) repertoire in response to the combination treatment and define functional responses to neoepitopes. These studies will enhance our understanding of the molecular mechanisms of tumor vaccination and facilitate the development of rational combination strategies.

Patterns and genomic correlates of PD-L1 expression in patients with biliary tract cancers.

Patients with biliary tract cancer (BTC) have a dismal prognosis and limited treatment options. Given the potential for immunotherapy in patients with BTC, we studied the expression of programmed death ligand-1 (PD-L1)/programmed death-1 (PD-1) and evaluated for associated genetic alterations in patients with BTC. By immunohistochemistry (IHC), PD-L1 (SP142 antibody; ≥2+ and/or ≥5% staining on tumor cells considered positive) and PD-1 [NAT105 antibody; ≥1+ staining of tumor infiltrating lymphocytes (TILs) considered positive] expression was studied and next-generation sequencing (NGS) was performed using Caris Life Sciences' sequencing panel of 592 genes. A total of 652 patients with BTC were included in this study: 77 extrahepatic cholangiocarcinoma (ECC), 203 gallbladder cancer (GBC), and 372 intrahepatic cholangiocarcinoma (ICC). Of the 652 tumors 8.6% were PD-L1 positive with the following distribution: GBC 12.3% (25/203), ICC 7.3% (27/372), and ECC 5.2% (4/77). There was a statistically significant increase in BRAF, BRCA2, RNF43, and TP53 mutations in PD-L1 positive group as compared to PD-L1 negative. Among other biomarkers tested, TOP2A, tumor mutational burden (TMB) high (≥17 mutations per megabase) (10.7%), and microsatellite instability high (MSI-H) (7.1%) were increased in PD-L1 positive tumors versus PD-L1 negative tumors. PD-L1 expression was noted in a small percentage (8.6%) of patients with BTC. This finding suggests potential benefit of immunotherapy in this subset of patients. Furthermore, there was a statistically significant association between PD-L1 expression and certain genomic alterations (BRAF, BRCA2, RNF43, TP53) and biomarkers (TOP2A, TMB high, MSI-H), which might direct the use of rational combination strategies and clinical trial development.

Ibrutinib and Venetoclax Target Distinct Subpopulation of CLL Cells: Rationale for Drug Combination and Implication of Minimal Residual Disease Eradication

Ibrutinib (Ibr) is a specific inhibitor of Bruton tyrosine kinase, a component of the B-cell receptor (BCR) signaling. Venetoclax (Veneto) inhibits the anti-apoptotic protein BCL2. Both drugs are highly effective as monotherapy against chronic lymphocytic leukemia (CLL) (Byrd NEJM, Roberts, NEJM, 2015 and Roberts, Blood, 2019) and clinical trials using the combination therapy are ongoing. An interesting clinical observation is that the tumor cell sensitivity to these two drugs differ between different anatomic compartments. While lymph node-resident CLL cells are more sensitive to ibr, circulating CLL cells in the peripheral blood are more sensitive to the action of veneto. When these two drugs are used in combination to treat relapsed/refractory (Hillman ASH 2018 and Kater EHA 2019) or previously untreated CLL patients (Jain NEJM 2019), rate of complete response significantly increases compared to single drug alone. Moreover, negativity of bone marrow minimal residual disease continues to improve over time. However, the reason behind these observed compartmental responses is largely unknown, and we investigated the differential response using an ex vivo model that promotes CLL proliferation (CLL proliferation model). A better understanding of how these two drugs synergize would eventually help develop other rational combination strategies. We established the ex vivo model using fibroblasts derived from the bone marrow of a CLL patient. In this model, CLL cells closely interact with the bone marrow fibroblasts (BMF) (Fig.1) and divide and proliferate for several generations, as measured by CFSE labeling. The culture, in some cases, may continue for 10 weeks before cell death ensues. With this ability to promote long-term cell proliferation, the model recapitulates one of the most salient features observed histologically in the "proliferation centers" of the CLL lymph nodes. With the proliferation model, we first tested the effects of ibrutinib on cell division/proliferation as well as cell viability in samples taken from 30 CLL patients consisting of 22 ibr-naïve (thus sensitive) and 8 ibr-relapsed (thus resistant) patients. We demonstrate that there was lack of a significant inhibition of ibr on cell viability. In comparison, Ibr markedly inhibited cell proliferation in cells from ibr-naïve/sensitive patients (p<0.0001), while such inhibition was much less prominent in cells from ibr-relapsed/resistant patients (p=0.053). Overall, our data demonstrate that only the dividing subpopulation of CLL responds to ibr, and this response is highly correlated with patients' clinical response to the drug while cell viability responses are not. With the CLL proliferation model, we also evaluated how veneto works. Veneto, as expected, markedly decreased cell viability at clinically achievable concentrations. CFSE profiles of the remaining live cells, however, revealed an interesting pattern of distribution, showing that veneto induces cell death but it preferentially kills the resting CLL population (Fig. 2). In other words, the resting subpopulation of CLL, instead of the dividing one, preferentially responds to veneto as compared to ibr. Combination of the two drugs, as expected, worked most effectively, significantly reducing the total number of live cells, both resting and dividing subpopulations, in all cases. Analyses of CFSE profiles became less meaningful in many of these cases due to the small number of live cells remaining after the combination treatment. In conclusion, our study, comparing the actions of ibr and veneto in a clinically relevant CLL proliferation model, demonstrates for the first time that the drugs target distinct subpopulations of CLL cells with different proliferative capacity. The data also suggest that single drug therapy may leave a subpopulation behind that would potentially rise to cause a future relapse. Our study provides a strong laboratory rationale explaining why combining these two drugs has the potential to eradicate CLL disease. The findings are also in line with the clinical observations regarding the compartmental response; ibrutinib works primarily on the lymph nodes where CLL cells divide in the "proliferation centers" and veneto acts preferentially on the circulating CLL cells that are mostly non-dividing and resting. Disclosures Wang: Incyte Reaserch Institute: Employment. Ma:Abbvie: Research Funding; Xeme: Research Funding; Novartis: Research Funding; Bioverativ: Consultancy; Janssen: Consultancy, Speakers Bureau; Beigene: Research Funding; Kite: Consultancy; Gilead: Research Funding; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Acerta: Research Funding; Astra Zeneca: Consultancy, Research Funding, Speakers Bureau; Genentech: Consultancy; Incyte: Research Funding; Juno: Research Funding. Larson:Celgene: Consultancy; Agios: Consultancy; Novartis: Honoraria, Other: Contracts for clinical trials.

EP1.04-09 A Cross-Sectional Study of Clinical Trials on Radiotherapy Combined with Immunotherapy for Lung Cancer

For patients with lung cancer, remarkable advances have been made in Immune-Oncology (IO) therapy, especially immune checkpoint inhibitors (ICI). However, multi-modality treatment is needed to improve the efficacy or enlarge the beneficial populations of IO treatment. This study is to comprehensively summarize and analyze the clinical trials focusing on radiotherapy (RT) combined with IO therapy, explore the trend of research, as well as provide a view of the latest landscape of combination strategies. Trials registered on the electronic database (https://clinicaltrials.gov) between Jan 2009 and Jan 2019 were searched using “Radiotherapy AND Immunotherapy | Recruiting, Not yet recruiting, Active, not recruiting, Completed, Enrolling by invitation Studies | Interventional Studies | Lung Cancer”. SPSS 20.0 was used to analyze the data. Totally 69 clinical trials of RT and IO combination therapy for lung cancer were recorded, including 54 active and 15 completed. Geographically, most of the trials were carried out in the USA (n=47, 68.1%), followed by the European countries (n=18, 26.1%). From timeline, the past 2 years has seen a soaring number of 40 clinical trials accounting for 58.0% of the total. The combination therapy trials were more often in non-small cell lung cancer (NSCLC) (n=52, 75.4%) than small cell lung cancer (SCLC) (n=10, 14.5%), with the remaining 7 trials unspecified. As for combination strategies, trials of tumor vaccine combined with RT were the most frequent before 2016. But at present, ICI has exceeded tumor vaccine in the combination with RT and makes up the absolute most (n=62, 89.9%). On the whole, most of the combination therapy trials are in phase I/II (n=64, 92.8%). Although most trials are set for advanced or metastatic cancers (n=43, 62.3%), there are a few exploring the safety and effectiveness of combination therapy for early stage cancers or as adjuvant therapy (n=9, 13.0%). One trial was set as neo-adjuvant therapy. As for RT details, 22 trials were SBRT combined with IO therapy. Two trials were exploring the optimal sequence of RT and IO therapy. And one trial is to compare high versus low dose RT in the combination with ICI in NSCLC. From the trials of RT and IO combination therapy for lung cancer, those of ICI combined with RT are increasing rapidly, although most are in phase I/II. Further studies are needed to explore the more detailed rational combination strategies, such as the sequencing, fractionation and dose of RT, and the optimal IO agent.

Targeting Autophagy by MPT0L145, a Highly Potent PIK3C3 Inhibitor, Provides Synergistic Interaction to Targeted or Chemotherapeutic Agents in Cancer Cells.

Anticancer therapies reportedly promote pro-survival autophagy in cancer cells that confers drug resistance, rationalizing the concept to combine autophagy inhibitors to increase their therapeutic potential. We previously identified that MPT0L145 is a PIK3C3/FGFR inhibitor that not only increases autophagosome formation due to fibroblast growth factor receptor (FGFR) inhibition but also perturbs autophagic flux via PIK3C3 inhibition in bladder cancer cells harboring FGFR activation. In this study, we hypothesized that combined-use of MPT0L145 with agents that induce pro-survival autophagy may provide synthetic lethality in cancer cells without FGFR activation. The results showed that MPT0L145 synergistically sensitizes anticancer effects of gefitinib and gemcitabine in non-small cell lung cancer A549 cells and pancreatic cancer PANC-1 cells, respectively. Mechanistically, drug combination increased incomplete autophagy due to impaired PIK3C3 function by MPT0L145 as evidenced by p62 accumulation and no additional apoptotic cell death was observed. Meanwhile, drug combination perturbed survival pathways and increased vacuolization and ROS production in cancer cells. In conclusion, the data suggest that halting pro-survival autophagy by targeting PIK3C3 with MPT0L145 significantly sensitizes cancer cells to targeted or chemotherapeutic agents, fostering rational combination strategies for cancer therapy in the future.

A Rational Combination Strategy Targeting ATM Kinase in Pediatric High-Grade Glioma

A Rational Combination Strategy Targeting ATM Kinase in Pediatric High-Grade Glioma

A Cross-Sectional Study of Clinical Trials on Radiotherapy Combined with Immunotherapy for Lung Cancer

For patients with lung cancer, remarkable advances have been made in Immune-Oncology (IO) therapy, especially immune checkpoint inhibitors (ICI). However, multi-modality treatment is needed to improve the efficacy or enlarge the beneficial populations of IO treatment. This study is to comprehensively summarize and analyze the clinical trials focusing on radiotherapy (RT) combined with IO therapy, explore the trend of research, as well as provide a view of the latest landscape of combination strategies. Trials registered on the electronic database (https://clinicaltrials.gov) between Jan.2009 and Jan.2019 were searched using “Radiotherapy AND Immunotherapy | Recruiting, Not yet recruiting, Active, not recruiting, Completed, Enrolling by invitation Studies | Interventional Studies | Lung Cancer”. Statistical analysis software was used to analyze the data. Totally 69 clinical trials of RT and IO combination therapy for lung cancer were recorded, including 54 active and 15 completed. Geographically, most of the trials were carried out in the USA (n=47, 68.1%), followed by the European countries (n=18, 26.1%). From timeline, the past 2 years has seen a soaring number of 40 clinical trials accounting for 58.0% of the total. The combination therapy trials were more often in non-small cell lung cancer (NSCLC) (n=52, 75.4%) than small cell lung cancer (SCLC) (n=10, 14.5%), with the remaining 7 trials unspecified. As for combination strategies, trials of tumor vaccine combined with RT were the most frequent before 2016. However, at present, ICI has exceeded tumor vaccine in the combination with RT and makes up the absolute most (n=62, 89.9%). On the whole, most of the combination therapy trials are in phase I/II (n=64, 92.8%). Although most trials are set for advanced or metastatic cancers (n=43, 62.3%), there are a few exploring the safety and effectiveness of combination therapy for early stage cancers or as adjuvant therapy (n=9, 13.0%). One trial was set as neo-adjuvant therapy. As for RT details, 22 trials were SBRT combined with IO therapy. Two trials were exploring the optimal sequence of RT and IO therapy. In addition, one trial is to compare high versus low dose RT in the combination with ICI in NSCLC. From the trials of RT and IO combination therapy for lung cancer, those of ICI combined with RT are increasing rapidly, although most are in phase I/II. Further studies are needed to explore the more detailed rational combination strategies, such as the sequencing, fractionation, and dose of RT, and the optimal IO agent.

HSP90/AXL/eIF4E-regulated unfolded protein response as an acquired vulnerability in drug-resistant KRAS-mutant lung cancer

Drug resistance and tumor heterogeneity are formidable challenges in cancer medicine, which is particularly relevant for KRAS-mutant cancers, the epitome of malignant tumors recalcitrant to targeted therapy efforts and first-line chemotherapy. In this study, we delineate that KRAS-mutant lung cancer cells resistant to pemetrexed (MTA) and anti-MEK drug trametinib acquire an exquisite dependency on endoplasmic reticulum (ER) stress signaling, rendering resistant cancer cells selectively susceptible to blockage of HSP90, the receptor tyrosine kinase AXL, the eukaryotic translation initiation factor 4E (eIF4E), and the unfolded protein response (UPR). Mechanistically, acquisition of drug resistance enables KRAS-mutant lung cancer cells to bypass canonical KRAS effectors but entail hyperactive AXL/eIF4E, increased protein turnover in the ER, and adaptive activation of an ER stress-relief UPR survival pathway whose integrity is maintained by HSP90. Notably, the unique dependency and sensitivity induced by drug resistance are applicable to KRAS-mutant lung cancer cells undergoing de novo intratumor heterogeneity. In line with these findings, HSP90 inhibitors synergistically enhance antitumor effects of MTA and trametinib, validating a rational combination strategy to treat KRAS-mutant lung cancer. Collectively, these results uncover collateral vulnerabilities co-occurring with drug resistance and tumor heterogeneity, informing novel therapeutic avenues for KRAS-mutant lung cancer.

Novel Apoptosis-Inducing Agents for the Treatment of Cancer, a New Arsenal in the Toolbox.

Evasion from apoptosis is an important hallmark of cancer cells. Alterations of apoptosis pathways are especially critical as they confer resistance to conventional anti-cancer therapeutics, e.g., chemotherapy, radiotherapy, and targeted therapeutics. Thus, successful induction of apoptosis using novel therapeutics may be a key strategy for preventing recurrence and metastasis. Inhibitors of anti-apoptotic molecules and enhancers of pro-apoptotic molecules are being actively developed for hematologic malignancies and solid tumors in particular over the last decade. However, due to the complicated apoptosis process caused by a multifaceted connection with cross-talk pathways, protein–protein interaction, and diverse resistance mechanisms, drug development within the category has been extremely challenging. Careful design and development of clinical trials incorporating predictive biomarkers along with novel apoptosis-inducing agents based on rational combination strategies are needed to ensure the successful development of these molecules. Here, we review the landscape of currently available direct apoptosis-targeting agents in clinical development for cancer treatment and update the related biomarker advancement to detect and validate the efficacy of apoptosis-targeted therapies, along with strategies to combine them with other agents.

Abstract 1322: Maximizing the therapeutic potential of SHP2 inhibition with rational combination strategies in tumors driven by aberrant RAS-MAPK signaling

Abstract SHP2 (PTPN11) is a protein tyrosine phosphatase and scaffold protein that functions as a convergent node downstream of multiple receptor tyrosine kinases (RTKs), integrating growth factor signals to promote RAS activation. Recently we reported that a SHP2 allosteric inhibitor suppresses RAS activation and proliferation in cancers driven by nucleotide cycling oncogenic KRAS, e.g., KRASG12C and other RAS-GTP dependent downstream mutations, e.g., NF1LOF, BRAFclass3. Daily oral administration of a SHP2 allosteric inhibitor at the maximally tolerated dose achieved tumor cytostasis in cell line-derived and patient-derived xenograft models harboring these mutations, and in some instances, tumor regressions. In most models, tumor cytostasis was followed by regrowth of tumors, reminiscent of patterns observed with MEK inhibitors. In the NCI-H358 KRASG12C model of non-small cell lung cancer, the maximum daily dose of the SHP2 inhibitor resulted in ~75% inhibition (relative to control) of pERK1/2, a downstream indicator of RAS-MAPK signaling in tumor lysates. These results support the hypothesis that targeting a single node results in incomplete RAS-MAPK pathway inhibition, which may permit tumor persistence in these models. To achieve deeper suppression of oncogenic flux through the RAS-MAPK pathway in tumors driven by nucleotide cycling KRAS, NF1LOF, or BRAFclass3, we evaluated combinations of MEK and ERK inhibitors with a SHP2 inhibitor as rational “in-pathway”, anti-proliferative combinations. Additionally, we examined the combination with CDK4/6 inhibition as a strategy to impact RAS-MAPK signaling both directly and via cell cycle effects. We found that concurrent targeting of SHP2 and MEK or ERK drives greater anti-tumor efficacy than that observed with monotherapy with either agent. Comparable synergy was observed when SHP2 inhibition was combined with CDK4/6 inhibition. Given the complementary anti-tumor mechanisms triggered by MEK/ERK and CDK4/6 inhibition, we further interrogated a triple combination of SHP2, MEK, and CDK4/6 inhibition to drive complete elimination of RAS-MAPK oncogenic signals. We found that this combination regimen was tolerated, resulted in more significant responses in all tumor models we tested, and delayed the evolution of on-treatment resistance. Investigation of the downstream effects of the triple therapy on tumor cells revealed profound combinatorial effects on cell cycle progression, proliferation and induction of apoptosis. These observed synergies are likely due to a combination of RAS pathway extinction and the inhibition of parallel escape mechanisms. In summary, our findings reveal rational combination strategies to maximize the therapeutic potential of a potent SHP2 inhibitor in patients with tumors harboring nucleotide cycling oncogenic KRAS or other RAS-GTP-dependent downstream mutations. Citation Format: Grace J. Lee, Carlos Stahlhut, James Evans, Denise F. Reyes, Edward G. Lorenzana, Shaoling Li, Elena S. Koltun, Dong Lee, Zhengping Wang, Robert J. Nichols, Jacqueline A. Smith, Mallika Singh. Maximizing the therapeutic potential of SHP2 inhibition with rational combination strategies in tumors driven by aberrant RAS-MAPK signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1322.