To systematically define molecular features in human tumour cells which determine their degree of sensitivity to human allogeneic natural killer (NK) cells, we quantified the NK cell responsiveness of hundreds of molecularly-annotated "DNA-barcoded" solid tumour cell lines in multiplexed format and applied genome-scale CRISPR-based gene editing screens in several solid tumour cell lines to functionally interrogate which genes in tumour cells regulate the response to NK cells. In these orthogonal studies, NK-sensitive tumour cells tend to exhibit "mesenchymal-like" transcriptional programs; high transcriptional signature for chromatin remodeling complexes; high levels of B7-H6 (NCR3LG1); low levels of HLA-E/antigen presentation genes. Importantly, transcriptional signatures of NK cell-sensitive tumour cells correlate with immune checkpoint inhibitor (ICI) resistance in clinical samples. This study provides a comprehensive map of mechanisms regulating tumour cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies.

The extent and importance of functional heterogeneity and crosstalk between tumor cells is poorly understood. Here, we describe the generation of clonal populations from a patient-derived ovarian clear cell carcinoma model which forms malignant ascites and solid peritoneal tumors upon intraperitoneal transplantation in mice. The clonal populations are engineered with secreted Gaussia luciferase to monitor tumor growth dynamics and tagged with a unique DNA barcode to track their fate in multiclonal mixtures during tumor progression. Only one clone, CL31, grows robustly, generating exclusively malignant ascites. However, multiclonal mixtures form large solid peritoneal metastases, populated almost entirely by CL31, suggesting that transient cooperative interclonal interactions are sufficient to promote metastasis of CL31. CL31 uniquely harbors ERBB2 amplification, and its acquired metastatic activity in clonal mixtures is dependent on transient exposure to amphiregulin, which is exclusively secreted by non-tumorigenic clones. Amphiregulin enhances CL31 mesothelial clearance, a prerequisite for metastasis. These findings demonstrate that transient, ostensibly innocuous tumor subpopulations can promote metastases via “hit-and-run” commensal interactions.

KB-0742 is an orally available, potent, and selective inhibitor of cyclin-dependent kinase 9 (CDK9). Sensitivity profiling across a panel of 800 adherent and suspension immortalized pan-cancer cell lines using the Broad Institute PRISM platform had previously demonstrated MYC amplification as being a driver of KB-0742 sensitivity. We sought to further determine the activity of KB-0742 in breast cancer cell lines, patient-derived cell and organoid cultures, and patient-derived xenograft (PDX) models. KB-0742 decreased the viability of cell lines derived from primary and metastatic breast cancers, including triple-negative breast cancers (TNBCs). In a panel of 17 breast cancer cell lines, including 2 human epidermal growth factor receptor 2-positive (HER2+), 7 estrogen-receptor positive (ER+), 1 HER2+/ER+ and 7 TNBCs, the effect of KB-0742 on viability was either cytostatic or cytotoxic over the duration of the culture period. KB-0742 was also tested in patient-derived cell lines derived from primary and metastatic tumors including TNBC, and in 2 TNBC MYC-high expressing patient-derived organoid cultures. KB-0742 demonstrated cytostatic and cytotoxic effects on cell growth and superior inhibitory effects on cell growth in the TNBC organoid cultures as compared with paclitaxel and gemcitabine. Since sensitivity to KB-0742 was observed in breast cancer cell lines and patient-derived models, including TNBCs, we evaluated efficacy and target engagement in several MYC-high expressing TNBC PDX animal models. Treatment with KB-0742 using an intermittent dosing schedule (3-days on/4-days off) was well tolerated and resulted in anti-tumor activity comparable to that observed with standard of care chemotherapeutic agents. Tumors collected at 2 and 8 hours post-final dose displayed a decrease in phosphorylated serine residue 2 (pSER2) in the C-terminal domain of RNA polymerase II (RNA pol-II) and decreased MYC protein levels consistent with CDK9 inhibition. These data demonstrate the efficacy of KB-0742 in preclinical models of breast cancer and supports clinical testing in TNBC patients. KB-0742 is currently being evaluated in a phase I dose-escalation trial in patients with relapsed or refractory solid tumors or non-Hodgkin lymphoma (NCT04718675). Citation Format: Douglas C. Saffran, Melinda A.L. Day, Nathalie Rioux, Tom Chen, Christina Lee, Suha Naffar-Abu Amara, David B. Freeman, Tressa Hood, Charles Y. Lin, Pavan Kumar, Jorge DiMartino. Preclinical activity of KB-0742, an oral, highly selective, CDK9 inhibitor, in cell lines and in MYC-high expressing, patient-derived models of multiple breast cancer subtypes [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-05.

Natural killer (NK) cells exhibit potent activity in pre-clinical models of diverse hematologic malignancies and solid tumors and infusion of high numbers of NK cells, either autologous or allogeneic, after their ex vivo expansion and activation, has been feasible and safe in clinical studies. To systematically define molecular features in human tumor cells which determine their degree of sensitivity to human allogeneic NK cells, we quantified the NK cell responsiveness of hundreds of molecularly-annotated “DNA-barcoded” solid tumor cell lines in multiplexed format (PRISM; Profiling Relative Inhibition Simultaneously in Mixtures approach), correlating cytotoxicity scores for each cell line with the CCLE transcriptional data (RNA-seq), to reveal genes that are associated with resistance or sensitivity to NK cells. In addition, we applied genome-scale CRISPR-based gene editing screens in several solid tumor cell lines to interrogate, at a functional level, which genes regulate tumor cell response to NK cells. Based on these orthogonal studies, NK sensitive tumor cells tend to exhibit high levels of the NK cell-activating ligand B7-H6 (NCR3LG1); low levels of the inhibitory ligand HLA-E; microsatellite instability (MSI) status; high transcriptional signature for chromatin remodeling complexes and low antigen presentation machinery genes. Treatment with an HDAC inhibitor reduced the sensitivity of SW620 colon cancer cells, increased antigen presentation machinery, including HLA-E, and reduced B7-H6. Importantly, we observe that transcriptional signatures of NK cell-sensitive tumor cells correlate with immune checkpoint inhibitor resistance in clinical samples. Strikingly, comprehensive analysis of the CCLE transcriptional signatures revealed that cell lines with mesenchymal-like program tend to be more sensitive to NK cells treatment, compared with cell lines of epithelial-like program. Indeed, mesenchymal tumors tend to have lower expression of antigen presentation machinery in both CCLE and TCGA, suggesting a link between these two machieneries. This study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies. Citation Format: Michal Sheffer, Emily Lowry, Nicky Beelen, Minasri Borah, Suha Naffar-Abu Amara, Chris C. Mader, Jennifer Roth, Aviad Tsherniak, Olga Dashevsky, Sara Gandolfi, Samantha Bender, Jordan Bryan, Cong Zhu, Li Wang, Ricardo De-Matos Simoes, Channing Yu, Yiguo Hu, Olli Dufva, Marios Giannakis, Todd Golub, Rizwan Romee, Satu Mustjoki, Aedin C. Culhane, Lotte Wieten, Constantine S. Mitsiades. Landscape of molecular events regulating tumor cell responses to natural killer cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO041.

To interrogate functional heterogeneity and crosstalk between tumor cells, we generated clonal populations from a patient-derived ovarian clear cell carcinoma model which forms malignant ascites and solid peritoneal tumors upon intraperitoneal transplantation in mice. The clonal populations were engineered with secreted Gaussia luciferase to monitor tumor growth dynamics and tagged with a unique DNA barcode to track their fate in multiclonal mixtures during tumor progression. Only one clone, CL31, grew robustly, generating exclusively malignant ascites. However, multiclonal mixtures formed large solid peritoneal metastases, populated almost entirely by CL31, suggesting that transient cooperative interclonal interactions were sufficient to promote metastasis of CL31. CL31 uniquely harbored ERBB2 amplification, and its acquired metastatic trait was dependent on transient exposure to amphiregulin, which was exclusively secreted by non-tumorigenic clones. Amphiregulin enhanced CL31 mesothelial clearance, a prerequisite for metastasis. These findings demonstrate that transient, ostensibly innocuous tumor subpopulations can promote metastases via "hitand-run" commensal interactions.

Liquid biopsies analyzing cell-free DNA (cfDNA) released by tumors are emerging as a powerful noninvasive sampling approach. As opposed to single-site solid tumor biopsy, liquid biopsies allow for better representation of the tumor mutational landscape. In the clinic, liquid biopsies are utilized for early detection of disease, to help inform treatment decisions, to monitor for disease recurrence, and to identify potential resistance mechanisms. Here we describe our approach to developing a liquid biopsy assay that will allow us to monitor, in real time, longitudinal changes in mutations’ allelic frequency in patients with solid tumors. Our approach combines two powerful technologies: next-generation sequencing, that enables the identification of a broad array of mutations, and droplet digital PCR (ddPCR) that provides speed and a cost-effective advantage to monitor mutations in cfDNA obtained from patient plasma samples. In order to maximize the effectiveness of using liquid biopsy assays in our clinical trial, we prioritized developing an approach that enabled us to meet the following criteria: 1) generating a baseline and end-of-treatment mutational profile for each patient using either a tumor tissue sample or liquid biopsy to identify both driver and potential resistance mutation markers, 2) longitudinal sampling and real-time analysis over the course of treatment, 3) high assay sensitivity to detect low frequency alleles, 4) overall cost-effectiveness, and 5) ability to examine allelic frequency of multiple mutations of interest from limited input of cfDNA isolated from plasma. Assay development included custom quantification of cfDNA and optimization of a pre-amplification step, which amplified the material allowing us to analyze multiple mutations of interest while ensuring that the quantitative nature of the assay was not impacted. For each individual mutation, gBlocks™ and/or cell-line−derived cfDNA were utilized to optimize annealing temperature and time, cycle number, and input amount to attain an assay with a target 0.1% limit of detection. These customized mutation assays can potentially serve as an accelerated cost-effective surrogate approach, compared to imaging, to monitor in real time the response to KB-0742 (CDK9i) in the ongoing phase 1/2 clinical trial NCT04718675. Citation Format: Suha Naffar-Abu Amara, Christina Noe, Tressa Hood, Sara Paganini, Crystal MacKenzie, Melinda A. Day, Pavan Kumar. Development of a liquid biopsy assay to longitudinally monitor changes in mutations’ allelic frequency in response to treatment with KB-0742 [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 535.

One of the challenging features of cancer is intratumoral heterogeneity (ITH). The existence of ITH can have profound consequences on tumor progression and drug sensitivity. However it is been essentially impossible to monitor the extent to which individual clonal populations contribute to and affect tumor behavior because of our inability to culture tumor cells from patients in vitro without loosing initial heterogeneity resulting from clonal selection under culture conditions. Through our collaboration with Dr. Tan Ince, we have access to cell culture medium (WIT-OC media) that allows primary tumor cells to undergo continuous population doublings without selecting for or against specific clones under culture conditions. By utilizing this media we have generated 80 single cell clones from a Clear cell Carcinoma (CCC) patient sample and monitored the extent of phenotypic and genomic heterogeneity among 12 of the clonal populations. These clones showed striking heterogeneity in copy number alterations as well as phenotypic heterogeneity in vitro as assessed by anoikis, growth in soft agar, doubling time, and growth in 3D reconstituted basement membrane cultures. We also examined correlations between in vitro properties and tumor formation in vivo using clones tagged with Gaussia-Luciferase (Gluc), a secreted luciferase that is secreted from cells and can be assayed in whole blood. We found that only three clones were transplantable by themselves; however, the clone which showed the most rapid expansion in the mouse was limited in it tumorigenic capacity, generating only ascitic tumor populations. To address whether clonal heterogeneity could contribute to tumor progression, we transplanted mixtures of clones and utilized barcoded clones to analyze the clonal composition of tumor masses. Interestingly, mixtures of the 12 clones resulted in the generation of more aggressive tumors, including the formation of solid tumors and metastases to lungs, liver and brain and allow in vivo growth; these metastatic growths contain distinct mixtures of individual clonal populations. Moreover, clones that could not initiate tumor formation individually could generate metastatic tumors, suggesting that there is cooperativity between the clones to enhance growth and induce metastasis. The study has provided evidence that cell lines generated from individual clones vary significantly in their functional activities in vitro and in vivo. Moreover, by studying the single cell clones, we have found evidence for the existence of interclonal crosstalk during tumor progression and are currently studying the nature and impact of such crosstalk on tumor progression and metastasis. Citation Format: Suha Naffar-Abu Amara, Laura Selfors, Marit Krohn, Tan Ince, Gordon Mills, Joan Brugge. Intratumoral cellular heterogeneity of epithelial ovarian carcinoma and its impact on tumor behavior. [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 2976. doi:10.1158/1538-7445.AM2014-2976

One of the most challenging aspects of cancer treatment is intratumoral heterogeneity (ITH). However it is been extremely difficult to monitor the extent to which individual clonal populations contribute to and affect tumor behavior because of the inability to culture tumor cells from patients in vitro without clonal selection under standard culture conditions, which lead to a loss in initial heterogeneity. Using a medium developed by Dr. Tan Ince that allows primary tumor cells to undergo continuous population doublings with minimal, if any, selection for or against specific clones, we generated 80 clonal populations from a Clear Cell Carcinoma (CCC) patient sample and monitored the extent of phenotypic and genomic heterogeneity among 12 of the clonal populations. These clones showed striking heterogeneity in copy number alterations as well as morphologic and phenotypic heterogeneity in vitro in assays such as anoikis, growth in soft agar, population doubling time, and growth in 3D reconstituted basement membrane cultures. To examine correlations between in vitro properties and tumor formation in vivo, we tagged the clones with Gaussia-Luciferase (Gluc), a secreted luciferase, which allowed us to monitor tumor burden over time using a blood-based assay. Based on tumor growth at 10 weeks, only two clonal populations were able to grow in the peritoneum when injected alone, one of which (#31) showed a much more rapid growth rate than the other; however #31 was still limited in its tumorigenic capacity, generating only ascitic tumor populations. To address whether functional crosstalk between clones affects tumor progression, we injected mixtures of the clones. Interestingly, mixtures of the 12 clones resulted in the generation of more aggressive in vivo phenotypes, e.g. the formation of solid peritoneal tumors and metastasis to lungs and brain, behaviors not detected with any individual clones. These results suggest that there is cooperativity between the clones to enhance growth and induce metastasis. The study has provided evidence that cell lines generated from individual clones vary significantly in their functional activities in vitro and in vivo. Moreover, by studying the single cell clones, we have found evidence for the existence of interclonal crosstalk during tumor progression and are currently studying the nature and impact of such cross talks on tumor progression and metastasis. This abstract is also presented as Poster A57. Citation Format: Suha Naffar-Abu Amara, Laura M. Selfors, Marit Krohn, Tan A. Ince, Bo R. Rueda, Rosemary Foster, Gordon B. Mills, Joan S. Brugge. Cellular heterogeneity of ovarian carcinoma cells and its impact on tumor behavior. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR09.