Supplemental Figures 1-6 and Supplemental Methods

Immunotherapy has revolutionized the way cancer is treated; however, despite tremendous success, not all patients benefit from immunotherapy. Identifying biomarkers predictive of effective treatment response to current and novel immunotherapies is of utmost importance. Multiple studies have indicated neutrophil-lymphocyte ratio (NLR) may be a predictive biomarker of immunotherapy response. We analyzed hematological parameters across multiple preclinical murine tumors to determine if NLR predicts tumor growth and response to treatment. The hematological survey identified NLR to be strongly correlated to tumor weights, with larger tumors exhibiting higher NLRs. In addition, mouse tumors that have been characterized as resistant or partially responsive to immunotherapy had a higher NLR than tumors responsive to immunotherapy. To track neutrophils in vivo, we generated a BAC transgenic mouse wherein firefly luciferase (Luc2), diphtheria toxin receptor (DTR), and enhanced green fluorescence protein (eGFP) genes were expressed under the endogenous Ly6G promoter. The Ly6G promoter enables tracking of Ly6G+ neutrophils using luciferase-based bioluminescence imaging (BLI), depletion of Ly6G+ neutrophils by diphtheria toxin treatment, and the ability to perform flow analyses using the eGFP marker. In vivo and ex vivo BLI demonstrated that the Ly6G+ cells were primarily expressed in bone marrow, blood, spleen, and the digestive tract. To ensure the specificity of the bioluminescence signal, we isolated Ly6G+ and Ly6G- immune cells and performed in vitro BLI. In vitro BLI confirmed that bioluminescence was exclusive to Ly6G+ cells. To confirm that the Luc2-DTR-eGFP (LDG) reporter cassette expression did not affect neutrophil activity and function, we quantified the level of myeloperoxidase (MPO), a marker of neutrophil activation, in the phorbol myristate acetate (PMA)-induced ear edema mouse model. Neutrophil activity was measured using luminol enabled BLI of MPO. Luminol-BLI following PMA-induced ear edema demonstrated similar MPO activity between wildtype and transgenic mice. Finally, we investigated if the LDG reporter cassette impacted tumor growth. Measures of tumor burden in this transgenic line recapitulated our hematological findings. Tumors with higher NLR showed higher tumor BLI signal when compared to tumors with lower NLR ratio, demonstrating that the LDG reporter cassette did not impact tumor growth in the transgenic mice and importantly, providing a tool to track Ly6G+ cells in tumors. Our results demonstrate that NLR in tumor correlates with response to immunotherapy across multiple tumor types. Here we describe the development of a transgenic mouse to track intratumoral Ly6G+ cells. These transgenic mice provide a valuable tool for profiling oncology compounds, including those targeting immune cells, and for understanding the mechanism of action of such agents. Citation Format: Milind D. Chalishazar, Nicolas Solban, Johnny Kopinja, Doug Linn, Razvan Cristescu, Heather Zhou, Thomas Rosahl, Brian Long, Weisheng Zhang, Eric Hostetler. Development of a bioluminescence reporter mouse model for tracking and quantifying Ly6G+ neutrophils in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2455.

MYC stimulates both metabolism and protein synthesis, but how cells coordinate these complementary programs is unknown. Previous work reported that, in a subset of small-cell lung cancer (SCLC) cell lines, MYC activates guanosine triphosphate (GTP) synthesis and results in sensitivity to inhibitors of the GTP synthesis enzyme inosine monophosphate dehydrogenase (IMPDH). Here, we demonstrated that primary MYChi human SCLC tumors also contained abundant guanosine nucleotides. We also found that elevated MYC in SCLCs with acquired chemoresistance rendered these otherwise recalcitrant tumors dependent on IMPDH. Unexpectedly, our data indicated that IMPDH linked the metabolic and protein synthesis outputs of oncogenic MYC. Coexpression analysis placed IMPDH within the MYC-driven ribosome program, and GTP depletion prevented RNA polymerase I (Pol I) from localizing to ribosomal DNA. Furthermore, the GTPases GPN1 and GPN3 were upregulated by MYC and directed Pol I to ribosomal DNA. Constitutively GTP-bound GPN1/3 mutants mitigated the effect of GTP depletion on Pol I, protecting chemoresistant SCLC cells from IMPDH inhibition. GTP therefore functioned as a metabolic gate tethering MYC-dependent ribosome biogenesis to nucleotide sufficiency through GPN1 and GPN3. IMPDH dependence is a targetable vulnerability in chemoresistant MYChi SCLC.

Small cell lung cancer (SCLC) is a neuroendocrine tumor treated clinically as a single disease with poor outcomes. Distinct SCLC molecular subtypes have been defined based on expression of ASCL1, NEUROD1, POU2F3, or YAP1. Here, we use mouse and human models with a time-series single-cell transcriptome analysis to reveal that MYC drives dynamic evolution of SCLC subtypes. In neuroendocrine cells, MYC activates Notch to dedifferentiate tumor cells, promoting a temporal shift in SCLC from ASCL1(+) to NEUROD1(+) to YAP1(+) states. MYC alternatively promotes POU2F3(+) tumors from a distinct cell type. Human SCLC exhibits intratumoral subtype heterogeneity, suggesting that this dynamic evolution occurs in patient tumors. These findings suggest that genetics, cell of origin, and tumor cell plasticity determine SCLC subtype.

Small-cell lung cancer (SCLC) is a highly aggressive neuroendocrine lung tumor that has been treated clinically as a homogeneous disease. Recent discoveries suggest that SCLC is heterogeneous with distinct molecular subtypes. Whether metabolic differences exist among SCLC subtypes is largely unexplored. We have aimed to determine whether metabolic vulnerabilities exist between SCLC subtypes that can be therapeutically exploited. Toward this end, we performed steady-state metabolomics on tumors isolated from distinct genetically engineered mouse models (GEMMs) representing the MYC and MYCL-driven subtypes of SCLC. We discovered that SCLC subtypes driven by different MYC family members have distinct metabolic profiles. Purine nucleotide biosynthesis and arginine/urea cycle pathways were enriched specifically in MYC-driven SCLC (Huang et al., Cell Metab 2108; Chalishazar et al., Clin Can Res 2019). MYC-driven SCLC preferentially depends on arginine-regulated pathways for polyamine biosynthesis and mTOR pathway activation. Chemoresistant SCLC cells exhibited increased MYC expression and similar metabolic liabilities as chemo-naive MYC-driven cells. Arginine depletion with pegylated arginine deiminase (ADI-PEG20) dramatically suppressed tumor growth and promoted survival of mice specifically with MYC-driven tumors, including in GEMMs, human cell line xenografts, and in new patient-derived xenograft (PDX) models. ADI-PEG20 was significantly more effective than the standard-of-care chemotherapy in GEMMs; however, tumors eventually relapse and acquire resistance to ADI-PEG20. Our current efforts are focused on identifying mechanisms of ADI-PEG20 resistance. We find that expression of the arginine biosynthetic enzyme ASS1 is frequently induced in ADI-PEG20 relapsed tumors in mouse and PDX models. Metabolite profiling of ADI-PEG20-resistant tumors suggests that ASS1 induction is associated with metabolic rewiring, which we predict will be associated with new metabolic vulnerabilities. Pathway analyses of metabolite data are consistent with the notion that ASS1 induction causes increased consumption of aspartate to generate arginine, and thereby ameliorate the demand for exogenous arginine. We predict that the diversion of aspartate away from nucleotide biosynthesis will lead to increased demand on other metabolic pathways for nucleotide biosynthesis. Preliminary data have identified pathways whose inhibition may cooperate with ADI-PEG20 to further extend the survival of mice with MYC-driven SCLC.

Abstract Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine tumor that is treated clinically as a single disease with poor outcomes. However, SCLC is recently recognized to comprise multiple molecular subsets with unique therapeutic vulnerabilities. Four distinct subtypes of SCLC have been defined based on expression of lineage-related transcription factors: ASCL1, NEUROD1, POU2F3 or YAP1. The origins of these subtypes remain unknown. We use mouse and human SCLC models with a time-series analysis of single-cell transcriptome profiling to reveal that the oncogene MYC drives the dynamic evolution of SCLC subtypes by activation of Notch signaling. MYC cooperates with Notch signaling to promote a temporal shift from an ASCL1-to-NEUROD1-to-YAP1-positive state from a neuroendocrine cell of origin, whereas MYC promotes POU2F3+ tumors from a distinct cell type. SCLC molecular subtypes are therefore not distinct, but rather represent dynamic stages of MYC-driven tumor evolution. Treatment-naive human SCLC exhibits intratumoral heterogeneity in SCLC subtypes, suggesting this dynamic evolution occurs in patient tumors. These findings demonstrate that genetics, cell of origin, and tumor cell plasticity determine SCLC subtype. Given the reported unique therapeutic vulnerabilities of each subtype, we postulate that SCLC tumors represent a “moving therapeutic target” that may require more general, combinatorial, or plasticity-directed therapeutic approaches to combat this transcriptional flexibility. We anticipate that molecular subsets of other cancer types may also represent dynamic stages of tumor evolution. Citation Format: Abbie S. Ireland, Alexi M. Micinski, David W. Kastner, Bingqian Guo, Sarah J. Wait, Kyle B. Spainhower, Christopher C. Conley, Opal S. Chen, Matthew R. Guthrie, Danny Soltero, Yi Qiao, Xiaomeng Huang, Szabolcs Tarapcsak, Siddhartha Devarakonda, Milind D. Chalishazar, Jason Gertz, Justin C. Moser, Gabor Marth, Sonam Puri, Benjamin L. Witt, Benjamin T. Spike, Trudy G. Oliver. MYC drives temporal evolution of small cell lung cancer subtypes by reprogramming neuroendocrine fate [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-120.

The natural history of small-cell lung cancer (SCLC) includes rapid evolution from chemosensitivity to chemoresistance, although mechanisms underlying this evolution remain obscure due to the scarcity of post-relapse tissue samples. We generated circulating tumor cell (CTC)-derived xenografts from patients with SCLC to study intratumoral heterogeneity (ITH) via single-cell RNA sequencing of chemosensitive and chemoresistant CTC-derived xenografts and patient CTCs. We found globally increased ITH, including heterogeneous expression of therapeutic targets and potential resistance pathways, such as epithelial-to-mesenchymal transition, between cellular subpopulations following treatment resistance. Similarly, serial profiling of patient CTCs directly from blood confirmed increased ITH post-relapse. These findings suggest that treatment resistance in SCLC is characterized by coexisting subpopulations of cells with heterogeneous gene expression leading to multiple, concurrent resistance mechanisms. These findings emphasize the need for clinical efforts to focus on rational combination therapies for treatment-naïve SCLC tumors to maximize initial responses and counteract the emergence of ITH and diverse resistance mechanisms. Stewart et al. use circulating tumor cell-derived xenografts from patients with small-cell lung cancer to study tumor heterogeneity following the onset of therapeutic resistance.

AbstractPurpose:Small-cell lung cancer (SCLC) has been treated clinically as a homogeneous disease, but recent discoveries suggest that SCLC is heterogeneous. Whether metabolic differences exist among SCLC subtypes is largely unexplored. In this study, we aimed to determine whether metabolic vulnerabilities exist between SCLC subtypes that can be therapeutically exploited.Experimental Design:We performed steady state metabolomics on tumors isolated from distinct genetically engineered mouse models (GEMM) representing the MYC- and MYCL-driven subtypes of SCLC. Using genetic and pharmacologic approaches, we validated our findings in chemo-naïve and -resistant human SCLC cell lines, multiple GEMMs, four human cell line xenografts, and four newly derived PDX models.Results:We discover that SCLC subtypes driven by different MYC family members have distinct metabolic profiles. MYC-driven SCLC preferentially depends on arginine-regulated pathways including polyamine biosynthesis and mTOR pathway activation. Chemo-resistant SCLC cells exhibit increased MYC expression and similar metabolic liabilities as chemo-naïve MYC-driven cells. Arginine depletion with pegylated arginine deiminase (ADI-PEG 20) dramatically suppresses tumor growth and promotes survival of mice specifically with MYC-driven tumors, including in GEMMs, human cell line xenografts, and a patient-derived xenograft from a relapsed patient. Finally, ADI-PEG 20 is significantly more effective than the standard-of-care chemotherapy.Conclusions:These data identify metabolic heterogeneity within SCLC and suggest arginine deprivation as a subtype-specific therapeutic vulnerability for MYC-driven SCLC.

MYC paralogs are frequently activated in small cell lung cancer (SCLC) but represent poor drug targets. Thus, a detailed mapping of MYC-paralog-specific vulnerabilities may help to develop effective therapies for SCLC patients. Using a unique cellular CRISPR activation model, we uncover that, in contrast to MYCN and MYCL, MYC represses BCL2 transcription via interaction with MIZ1 and DNMT3a. The resulting lack of BCL2 expression promotes sensitivity to cell cycle control inhibition and dependency on MCL1. Furthermore, MYC activation leads to heightened apoptotic priming, intrinsic genotoxic stress and susceptibility to DNA damage checkpoint inhibitors. Finally, combined AURK and CHK1 inhibition substantially prolongs the survival of mice bearing MYC-driven SCLC beyond that of combination chemotherapy. These analyses uncover MYC-paralog-specific regulation of the apoptotic machinery with implications for genotype-based selection of targeted therapeutics in SCLC patients.

Small cell lung cancer (SCLC) is a rapidly lethal disease with few therapeutic options. We studied metabolic heterogeneity in SCLC to identify subtype-selective vulnerabilities. Metabolomics in SCLC cell lines identified two groups correlating with high or low expression of the Achaete-scute homolog-1 (ASCL1) transcription factor (ASCL1(High) and ASCL1(Low)), a lineage oncogene. Guanosine nucleotides were elevated in ASCL1(Low) cells and tumors from genetically engineered mice. ASCL1(Low) tumors abundantly express the guanosine biosynthetic enzymes inosine monophosphate dehydrogenase-1 and -2 (IMPDH1 and IMPDH2). These enzymes are transcriptional targets of MYC, which is selectively overexpressed in ASCL1(Low) SCLC. IMPDH inhibition reduced RNA polymerase I-dependent expression of pre-ribosomal RNA and potently suppressed ASCL1(Low) cell growth in culture, selectively reduced growth of ASCL1(Low) xenografts, and combined with chemotherapy to improve survival in genetic mouse models of ASCL1(Low)/MYCHigh SCLC. The data define an SCLC subtype-selective vulnerability related to dependence on de novo guanosine nucleotide synthesis.

Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine lung tumor that has historically been treated as a single disease. Loss of the tumor suppressors RB1 and TP53, and amplifications inMYCfamily members, are frequent events in SCLC. We show that Myc expression cooperates withRb1 andTrp53loss in the mouse lung to promote aggressive, highly metastatic tumors, that are initially sensitive to chemotherapy followed by relapse, similar to human SCLC. Importantly, MYC drives a neuroendocrine-low ''variant'' subset of SCLC with low ASCL1 and high NEUROD1 expression corresponding to transcriptional profiles of human SCLC. Targeted drug screening reveals that SCLC with high MYC expression is vulnerable to Aurora kinase inhibition, which, combined with chemotherapy, strongly suppresses tumor progression and increases survival (Mollaoglu et al, Cancer Cell, 2017; Cardnell et al, Oncotarget, 2017). These findings were recently recapitulated in clinical trials where patients with relapsed SCLC exhibited significantly longer survival when treated with an Aurora kinase inhibitor specifically if tumors were high for MYC. More recently, our work and others suggests that MYC-high SCLC is also vulnerable to CHK1 inhibition. These data identify molecular features for patient stratification of SCLC and uncover a potential targeted treatment approach for MYC-driven SCLC. More recently, we have performed unbiased metabolic profiling of MYC-driven SCLC cell lines and murine tumors compared to MYCL and MYCN-driven samples. We find that MYC-driven SCLC is metabolically distinct in vitro and in vivo.Our findings reveal that guanosine nucleotides and biosynthetic enzyme Inosine Monophosphate Dehydrogenase-1 and -2 (IMPDH1 and IMPDH2) are elevated in MYC-high/ASCL1-low tumors and cell lines. IMPDH inhibition using Mizoribine selectively impeded the growth of MYC-high/ASCL1-low xenografts, and combined with chemotherapy to improve survival in MYC-driven genetically engineered mouse models (Huang et al, Cell Metabolism, In Press). These data strongly suggest that SCLC is composed of unique molecular subtypes with specific vulnerabilities to targeted therapy that should be considered in basic studies and clinical trial design. Cardnell RJ, Li L, Sen T, Bara R, Tong P, Fujimoto J, Ireland AS, Guthrie MR, Bheddah S, Banerjee U, Kalu NN, Fan YH, Dylla SJ, Johnson FM, Wistuba II, Oliver TG, Heymach JV, Glisson BS, Wang J, Byers LA (2017). Protein expression of TTF1 and cMYC define distinct molecular subgroups of small cell lung cancer with unique vulnerabilities to aurora kinase inhibition, DLL3 targeting, and other targeted therapies. Oncotarget, 8(43), 73419-73432. Huang F, Ni M, Chalishazar MD, Huffman KE, Kim J, Cai L, Shi X, Zacharias LG, Cai F, Gu W, Ireland AS, Gazdar AF, Oliver TG, Minna JD, Hu Z, and DeBerardinis RJ (In Press). Inosine monophosphate dehydrogenase dependence in a subset of small cell lung cancers. Cell Metab, 2018. Mollaoglu G, Guthrie MR, Bohm S, Bragelmann J, Can I, Ballieu PM, Marx A, George J, Heinen C, Chalishazar MD, Cheng H, Ireland AS, Denning KE, Mukhopadhyay A, Vahrenkamp JM, Berrett KC, Mosbruger TL, Wang J, Kohan JL, Salama ME, Witt BL, Peifer M, Thomas RK, Gertz J, Johnson JE, Gazdar AF, Wechsler-Reya RJ, Sos ML, Oliver TG (2017). MYC Drives Progression of Small Cell Lung Cancer to a Variant Neuroendocrine Subtype with Vulnerability to Aurora Kinase Inhibition. Cancer Cell, 31(2), 270-285. MYC, Aurora kinase, small cell lung cancer

Abstract Small cell lung cancer (SCLC) has largely been treated in the clinic as a homogeneous disease for the last 40 years. However, it is become increasingly appreciated that SCLC exhibits both intra- and intertumoral heterogeneity. Genetic loss of the tumor suppressors RB1 and TP53 is nearly universal in SCLC, while amplifications in MYC family members including C-, L-, and N-MYC are mutually exclusive. Using genetically engineered mouse models (GEMMs), we show that MycT58A expression cooperates with Rb1 and Trp53 loss in the mouse lung to promote aggressive, highly metastatic tumors that are initially sensitive to chemotherapy followed by relapse, similar to human SCLC. Importantly, MYC drives a neuroendocrine-low ‘‘variant’’ subset of SCLC with low ASCL1 and high NEUROD1 expression corresponding to transcriptional profiles of human SCLC. The MYC-driven subset of SCLC is also low for other clinically relevant biomarkers such as TTF1/NKX2.1 and DLL3. Targeted drug screening reveals that SCLC with high MYC expression is vulnerable to Aurora kinase inhibition, which, combined with chemotherapy, strongly suppresses tumor progression and increases survival. These findings are consistent with the results of recent clinical trials in patients with relapsed SCLC who received paclitaxel with or without the AURKA inhibitor alisertib. Furthermore, recent preclinical studies demonstrate that the MYC-driven subset of SCLC is preferentially sensitive to CHK1 inhibition and other metabolic targets compared to MYCL-driven SCLC. These data identify molecular features for patient stratification and uncover potential targeted treatment approaches for MYC-driven SCLC. Citation Format: Gurkan Mollaoglu, Matthew R. Guthrie, Stefanie Bohm, Johannes Bragelmann, Milind D. Chalishazar, Abbie S. Ireland, Fang Huang, Zeping Hu, Robert J. Cardnell, Triparna Sen, Jason Gertz, Jane E. Johnson, Adi F. Gazdar, Lauren A. Byers, Ralph J. DeBerardinis, Robert J. Wechsler-Reya, Martin Sos, Trudy G. Oliver. MYC drives molecular and therapeutically distinct subtype of SCLC [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr IA27.

Loss of the tumor suppressors RB1 and TP53 and MYC amplification are frequent oncogenic events in small cell lung cancer (SCLC). We show that Myc expression cooperates with Rb1 and Trp53 loss in the mouse lung to promote aggressive, highly metastatic tumors, that are initially sensitive to chemotherapy followed by relapse, similar to human SCLC. Importantly, MYC drives a neuroendocrine-low “variant” subset of SCLC with high NEUROD1 expression corresponding to transcriptional profiles of human SCLC. Targeted drug screening reveals that SCLC with high MYC expression is vulnerable to Aurora kinase inhibition, which, combined with chemotherapy, strongly suppresses tumor progression and increases survival. These data identify molecular features for patient stratification and uncover a potential targeted treatment approach for MYC-driven SCLC.

MM-398 is a stable nanotherapeutic encapsulation of the prodrug irinotecan (CPT-11) with longer plasma half-life and higher tumor deposition due to an enhanced permeability and retention effect. Pancreatic cancer has responded poorly to many therapeutics, largely because of inadequate drug penetration due to poor vascularization and the highly aggressive, hypoxic nature of the disease. We sought to better understand how MM-398, a relatively large (100nm) liposomal nanotherapeutic, could be used treat pancreatic cancer. We have tested MM-398 in several pancreatic xenograft models: BxPC3 (KRAS wild type), AsPC-1(KRAS G12D) , Panc-1 (KRAS G12D) and MiaPaCa (KRAS G12C). All models demonstrated complete tumor regression at 20 mg/kg or a human equivalent dose of 60-120 mg/m2. At this same dose, MM-398 suppresses tumor growth in a gemcitabine insensitive AsPC-1 xenograft. MM-398 functionally blocked AsPC-1 tumor cell proliferation as measured by ki-67 staining; however, gemcitabine administered at its maximum tolerated dose did not impact proliferation. MM-398 is currently in multiple clinical trials, including a phase 3 trial for patients with advanced gemcitabine-resistant pancreatic cancer (NAPOLI-1). In order to further understand mechanisms driving response to MM-398, we screened and ranked several cell lines for their ability to convert irinotecan into the active metabolite, SN38. BxPC3 and HT-29 tumors ranked highest in ability to convert irinotecan to SN-38, as measured by HPLC. In a BxPC3 pancreatic orthotopic model which spontaneously metastasizes, 10 mg/kg MM-398 significantly reduced both primary and metastatic tumor load as measured by ex vivo biophotonic imaging of BxPC3luc cells to spleen, lung, liver, diaphragm and GI associated lymph nodes (p In summary, MM-398 induces tumor regression in multiple mouse models of pancreatic cancer, including an orthotopic metastatic model. MM-398 activity may be driven in part by the ability to modify tumor microenvironment parameters, such as hypoxia and vascularization, both of which limit efficacy of chemotherapeutic agents in the treatment of pancreatic cancer. These data support the continued investigation of MM-398 in pancreatic cancer. Citation Format: Nancy Paz, Peter Laivins, Clet Niyikiza, Ulrik Nielsen, Jonathan Fitzgerald, Ashish Kalra, Milind Chalishazar, Stephan Klinz, Jaeyeon Kim, Daryl Drummond, Dmitri Kirpotin, Victor Moyo, Eliel Bayever. MM-398/PEP02, a novel liposomal formulation of irinotecan, demonstrates stromal-modifying anticancer properties. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A63.

Abstract MM-398 is a stable nanotherapeutic encapsulation of the prodrug irinotecan with an extended plasma half-life and higher intratumoral deposition compared with free-irinotecan. MM-398 is currently in multiple clinical trials, including a phase 3 trial for patients with advanced gemcitabine-resistant pancreatic cancer (NAPOLI-1). Pancreatic cancer has been described as being notoriously difficult to treat, potentially due to inadequate drug penetration through the dense stroma, or because the hypoxic tumor microenvironment suppresses cytotoxic activity. We sought to better understand how MM-398, a relatively large (100nm) liposomal nanotherapeutic, could potentially treat pancreatic cancer by determining the relative roles of systemic vs. local tumor activation of irinotecan in contributing to the activity of MM-398. Using a systems pharmacology approach, we developed a mechanistic pharmacokinetic (PK) model of MM-398 and free-irinotecan to predict both plasma and intratumoral levels of irinotecan and SN-38. The model was trained with PK and biodistribution data from mice bearing HT-29 xenografts, which were administered intravenously with varying doses of MM-398 or free-irinotecan. Model simulations predicted that MM-398 resulted in equivalent SN-38 exposure (area under curve, AUC) in tumor at a fivefold lower dose than free-irinotecan. However, an in vivo animal activity study showed that 15-fold lower dose of MM-398 was sufficient to yield equal growth inhibition of HT-29 xenografts, which reveals the limit of relating simple AUC-based exposure to in vivo tumor response. While intratumoral SN-38 exposure from free-irinotecan was limited to the first 48 hours after dosing, MM-398 maintained high levels of SN-38 throughout the week-long time window. Further analysis of the exposure-response identified that the duration of intratumoral SN-38 levels above the threshold was a valid predictive marker for xenograft tumor response. Identifying the source of intratumoral SN38 is confounded by the fact that the mouse species has an additional carboxylesterase (CES) that can convert irinotecan to SN-38 in serum. The serum SN-38/irinotecan ratio in mice is tenfold higher than that observed in humans. In order to translate this preclinical observation into the clinic, it is critical to identify the role of mouse-specific serum CES on intratumoral SN-38 exposure. Thus, we performed a PK study with knockout mice lacking the Ces1c gene, which encodes serum CES, and then retrained our mechanistic PK model. Serum SN-38 levels in the Ces1c knockout mice were measurably decreased by ˜85% in the central compartment. In contrast, simulating the effect of knock-out of either serum CES or tumor CES, predicts that the duration of intratumoral residence of SN-38 is significantly affected by tumor CES, rather than serum CES. This suggests that local activation to SN-38 by tumor CES as the main driver for SN-38 tumor residence, which in turn drives response. In summary, we applied a systems pharmacology approach to identify the importance of tumor CES (local SN-38 generation) as one of the determinants of MM-398 response. Liposomal encapsulation of irinotecan dramatically alters the pharmacokinetic profile of SN-38 in the tumor, as well as tumor response, by maintaining SN-38 levels above the response threshold. Local, sustained activity of this active irinotecan metabolite could result in prolonged cytotoxic and tumor microenvironment modifications with beneficial effects on treatment of pancreatic cancer and other solid tumors. Citation Format: Jaeyeon Kim, Eliel Bayever, Peter Laivins, Clet Niyikiza, Ulrik Nielsen, Jonathan Fitzgerald, Ashish Kalra, Milind Chalishazar, Stephan Klinz, Nancy Paz, Bart Hendriks, Daryl Drummond, Dmitri Kirpotin, Victor Moyo. Sustained intratumoral activation of MM-398 results in superior activity over irinotecan demonstrated by using a systems pharmacology approach [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr A6.

MM-398 is a stable nanotherapeutic encapsulation of the pro-drug irinotecan (CPT-11). Irinotecan, a topoisomerase inhibitor is currently being used in clinical practice for treatment of several indications; however, associated toxicities, mainly, neutropenia and gastrointestinal toxicity, have limited its clinical utility. Previously, we have demonstrated that MM-398 treatment resulted in significantly higher intratumor concentrations of both irinotecan (142-fold) and SN-38 (9-fold), thereby exhibiting enhanced anti-tumor activity compared to free irinotecan in different xenograft models. Multiple phase 1 and 2 studies have established a pharmacokinetic and safety profile. Recent data support continued clinical development for the drug in various indications, including pancreatic, gastric, colorectal and potentially other solid tumors. In order to further understand the enhanced anti-tumor activity of MM-398 we developed a mechanism-based PK model of MM-398 and free irinotecan designed to predict intratumor levels of SN-38. Based on this model, we evaluated the role of various determinants of response of MM-398. Sensitivity analysis revealed that the local activation of MM-398 was important for obtaining higher SN-38 intratumor levels compared to free irinotecan. To identify cell types responsible for local activation of MM-398 we investigated cellular liposome phagocytosis ability. In vitro studies demonstrated preferential uptake of MM-398 by phagocytic macrophages compared to tumor cells. FACS analysis of tumor samples (from subcutaneous xenografts) highlighted higher uptake of labeled liposomes by CD11b + and F4/80+ cells as compared to tumor cells. We are developing in vivo systems using engineered cell lines overexpressing growth factors to recruit macrophages (CSF1) or overexpressing irinotecan conversion enzymes to further investigate the role of macrophagesin local tumor conversion of MM-398 and to validate the model prediction that local activation is critical to MM-398 mechanism of action. The extent of tumor vascularization and permeability were also highlighted in the sensitivity analysis. To determine the effect of MM-398 on these parameters, we treated mice bearing HT29 (colorectal cancer) xenografts with a single dose of MM-398 and measured hypoxic markers (CAIX) and microvessel density (CD31) by immunohistochemistry. Tumors treated with MM-398 showed a greater degree of CD31 staining and lower CAIX staining, indicating that MM-398 may be able to affect the tumor microenvironment. We are currently evaluating how the ability of MM-398 to alter the tumor microenvironment affects the activity of other chemotherapeutic agents in combination therapy. These findings could support the use of MM-398 as a combination modality against tumors that may have acquired resistance to traditional chemotherapeutic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5696. doi:1538-7445.AM2012-5696