Targeting PIK3CA-mutant colorectal cancers with precision medicine strategies is of great clinical interest. However, resistance to single-agent PI3K pathway inhibitors has been observed across multiple clinical trials, necessitating the identification of combination therapies that overcome or prevent resistance to precision medicine strategies. Previously, our group identified that inhibition of mTORC1/2 is necessary to induce a response in PIK3CA-mutant colorectal cancers. The PI3K/mTORC1/2 inhibitor copanlisib has demonstrated some clinical activity in PIK3CA-mutant solid tumors as part of the NCI-MATCH trial. In this study, we evaluate potential combination therapies that could enhance the efficacy of copanlisib and other similar inhibitors in PIK3CA-mutant colorectal cancers. Using a novel high-throughput drug screen method in Apc- and Pik3ca-mutant mouse-derived cancer organoids, we identify navitoclax, a BCL-2 family inhibitor, as a drug that could potentially enhance the response to copanlisib. Across multiple in vitro and in vivo colorectal cancer models, navitoclax enhanced PI3K/mTOR inhibition (copanlisib, sapanisertib, and dactolisib) and induced apoptosis. Furthermore, we examine these combination therapies across a panel of patient-derived cancer organoids with a range of mutation profiles. These studies indicate that KRAS mutations could confer resistance. Furthermore, we identify BCL-xL as the major BCL-2 family target important for the response to this combination in this setting. This provides a strong rationale for mTORC1/2 and BCL-2 family inhibition as a potential treatment strategy for PIK3CA-mutant colorectal cancers.

Tumor heterogeneity is predicted to confer inferior clinical outcomes with precision-based strategies, however, modeling heterogeneity in a manner that still represents the tumor of origin remains a formidable challenge. Sequencing technologies are limited in their ability to identify rare subclonal populations and predict response to treatments for patients. Patient-derived organotypic cultures have significantly improved the modeling of cancer biology by faithfully representing the molecular features of primary malignant tissues. Patient-derived cancer organoid (PCO) cultures contain subclonal populations with the potential to recapitulate heterogeneity, although treatment response assessments commonly ignore diversity in the molecular profile or treatment response. Here, we demonstrate the advantage of evaluating individual PCO heterogeneity to enhance the sensitivity of these assays for predicting clinical response. Additionally, organoid subcultures identify subclonal populations with altered treatment response. Finally, dose escalation studies of PCOs to targeted anti-EGFR therapy are utilized which reveal divergent pathway expression when compared to pretreatment cultures. Overall, these studies demonstrate the importance of population-based organoid response assessments, the use of PCOs to identify molecular heterogeneity not observed with bulk tumor sequencing, and PCO heterogeneity for understanding therapeutic resistance mechanisms.

Abstract Rapid developments in cancer genetics and hereditary cancer risk assessments have enabled the identification of individuals at elevated risk for hereditary malignancies to guide enhanced cancer screening and prevention efforts. Multigene panel testing has emerged as the standard approach as emphasized in the National Comprehensive Cancer Network (NCCN) guidelines. The purpose of this study was to investigate the frequency of other pathogenic mutations identified through multigene panel testing in individuals who previously tested negative for germline BRCA1/2 and assess the clinical impact of panel testing. Patients either previously diagnosed with breast cancer or with a family history of breast or ovarian cancer that had also been seen by Oncology Genetics at a single institution and tested negative for BRCA1/2 genes but had not undergone prior multigene panel testing were invited via a letter to return for an additional genetic counseling visit to discuss multigene panel testing. Patients were also able to opt-in to a survey component of the study and received an email to fill out the Multidimensional Impact of Cancer Risk Assessment (MICRA) and Cancer Worry Scale (CWS). Surveys were completed prior to the initial genetic counseling appointment and repeated after discussion of the multigene panel test results. A total 24 women met with a genetic counselor of which 22 (92%) had a personal history of breast cancer, 1 (4%) had a family history of breast cancer, and 1 (4%) had a family history of ovarian cancer. The mean age was 52.6 years (range, 42-68) and 100% of women were Caucasian. Of the 24 women, 17 (71%) completed multigene panel testing. Of the women that underwent testing, 7 (41%) had negative results, 6 (35%) had variants of unknown significance (VUS), and 4 (24%) had a pathogenic mutation identified in another gene (CDKN2A, CHEK2, and CFTR). There was no significant change in MICRA or CWS scores after multigene panel results were reviewed in the 9 patients that completed both pre- and post-genetic testing surveys. The mean/median summary MICRA scores pre- and post-counselling were 30 (SD 12.4), median 26 (min 18, max 56), and 21.2 (SD 15.4), median 21 (min 3, max 43), respectively (p=0.19, Wilcoxon signed ranks test). The mean/median summary CWS scores pre- and post-counselling were 14.3 (SD 5), median 15 (min 7, max 24), and 13.6 (SD 2.59), median 14 (min 10, max 17), respectively (p=0.53, Wilcoxon signed ranks test). Of the 4 patients with newly identified pathogenic mutations, 2 started new screening protocols and 1 underwent a prophylactic mastectomy. In conclusion, multigene panel testing identified pathogenic mutations in a subset of individuals who previously tested negative for BRCA1/2 genes leading to changes in clinical management. The completion of multigene panel testing did not lead to a significant increase in MICRA or CWS scores. Key limitations of this study include a small sample size and an all Caucasian population. Citation Format: Devon Miller, Stephanie Pritzl, Angela Tess, Jessica Gooding, Lisa Barroilhet, Lori Dubenske, Kari Wisinski. Clinical Impact of Multigene Panel Testing in Patients with a Personal or Family History of Breast or Ovarian Cancer Previously Negative for BRCA1/2 Genes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-09-03.

Gastroesophageal cancers are highly diverse tumors in terms of their anatomic and molecular characteristics, making drug development challenging. Recent advancements in understanding the molecular profiles of these cancers have led to the identification of several new biomarkers. Ongoing clinical trials are investigating new targeted agents with promising results. CLDN18.2 has emerged as a biomarker with established activity of associated targeted therapies. Other targeted agents, such as bemarituzumab and DKN-01, are under active investigation. As new agents are incorporated into the treatment continuum, the questions of biomarker overlap, tumor heterogeneity, and toxicity management will need to be addressed.

Purpose Biomarker-based therapies have shown improved patient outcomes across various cancer types. The purpose of this review to summarize our knowledge of current and future biomarkers in esophagogastric adenocarcinoma (EGA). Methods In this publication, we will review current standard biomarkers in patients with upper GI cancers. We will also discuss novel biomarkers that are under investigations and their associated therapies that are currently in clinical trials. Results EGAa are a group of heterogeneous diseases, both anatomically and molecularly. There are several established biomarkers (HER2, PD-L1, microsattelite instability or mismatch repair protein expression) that allow for individualized treatments for patients with these cancers. There are also several emerging biomarkers for EGA, some of which have clinically relevant associated therapies. Claudin 18.2 is the furthest along among these. Anti-claudin antibody, zolbetuximab, improved overall survival in biomarker select patients with advanced GEA in two phase 3 studies. Other novel biomarkers, such as FGFR2b and DKN01, are also in the process of validation, and treatments based on the presence of these biomarkers are currently in clinical studies. Conclusion Ongoing efforts to identify novel biomarkers in EGA have led to enhanced subclassification of upper GI cancers. These advances, coupled with the strategic application of targeted therapies and immunotherapy when appropriate, hold promise to further improve patients outcomes.

(A) Individual replicates of data shown in Figure 6A. (B) Individual replicates of data from Figure 6D.

Success rates for growing human cancers in spheroid culture

Detailed characteristics of CRC PDOCS.

AbstractBackgroundSarcomas are rare diagnoses but are seen with relative frequency in adolescents and young adults and thus can present in pregnancy. We sought to study the administration of anthracyclines and/or ifosfamide in pregnancy‐associated sarcomas.Patients and MethodsWe conducted a multi‐institutional retrospective study, identifying sarcoma patients who received anthracyclines and/or ifosfamide during pregnancy. Chart review identified variables related to demographics, cancer diagnosis, therapies, and outcome of the patient and fetus. Wilcoxon rank‐sum test compared two independent samples.ResultsWe identified 13 patients at seven institutions with sarcoma who received anthracyclines and/or ifosfamide during pregnancy, including four bone sarcomas and nine soft tissue sarcomas diagnosed at a mean gestational age of 16.7 ± 5.9 weeks. Only nine patients had live births (9/13, 69.2%), with mean gestational age of 30.8 ± 3.8 weeks at delivery. The four patients with pregnancy loss all received both doxorubicin and ifosfamide, with chemotherapy initiated at 15.5 weeks as compared with 21.3 weeks for those patients with live births (p = 0.016).ConclusionIn this multi‐institutional study of sarcoma chemotherapy regimens administered during pregnancy, we found a high rate of fetal demise that was seen only in patients receiving both doxorubicin and ifosfamide and statistically more likely with chemotherapy initiation earlier in the second trimester. While limited by a small sample size, our study represents the largest study of sarcoma patients that received anthracyclines and/or ifosfamide in pregnancy thus far reported and supports development of an international registry to study concerns raised by our study.

Purpose Estimating cancer risk associated with interplanetary space travel is complicated. Human exposure data to high atomic number, high-energy (HZE) radiation is lacking, so data from low linear energy transfer (low-LET) gamma-ray radiation is used in risk models, with the assumption that HZE and gamma-ray radiation have comparable biological effects. This assumption has been challenged by reports indicating that HZE radiation might produce more aggressive tumors. The goal of this research is to test whether high-LET HZE radiation induced tumors are more aggressive. Materials and methods Murine models of mammary and liver cancer were used to compare the impact of exposure to 0.2Gy of 300MeV/n silicon ions, 3 Gy of gamma-rays or no radiation. Numerous measures of tumor aggressiveness were assessed. Results For the mammary cancer models, there was no significant change in the tumor latency or metastasis in silicon-irradiated mice compared to controls. For the liver cancer models, we observed an increase in tumor incidence but not tumor aggressiveness in irradiated mice. Conclusion Tumors in the HZE-irradiated mice were not more aggressive than those arising from exposure to low-LET gamma-rays or spontaneously. Thus, enhanced aggressiveness does not appear to be a uniform characteristic of all tumors in HZE-irradiated animals.

Abstract Colorectal cancer (CRC) is a leading cause of cancer-related death. PIK3CA mutations are common, leading to a constitutively active phosphoinositide-3 kinase (PI3K). An effective means to target this pathway has yet to be identified. We investigated the use of a panel of inhibitors targeting the PI3K pathway including copanlisib (dual PI3K/mTOR), BYL-719 (alpha isomer specific PI3K), GDC-0941 (pan PI3K), and TAK-228 (MTORC1/2). To test the efficacy of these inhibitors in CRC, murine organotypic cancer spheroids (MDOCS) were generated from the invasive adenocarcinomas of Apc and Pik3ca transgenic mice. These inhibitors were investigated at clinically relevant doses (100-400nM). Copanlisib and TAK-228 were the only inhibitors to result in a significant reduction in the size of the MDOCS (200nM; 27% p-value<0.001, 18% p-value<0.001, respectively). This result correlated with a decrease in the phosphorylation of AKT (ser473), RPS6, and 4EBP1. Minimal induction of apoptosis was observed using these inhibitors alone as measured by cleaved PARP and cleaved caspase 3. These results were confirmed in vivo using transgenic mice with TAK-228 (1mg/kg/day) and copanlisib (10mg/kg q2d x5) resulting in a reduction in lumen occlusion of the colon tumors. Persistent BCL-2 and BCL-xL signaling was hypothesized to be preventing the induction of apoptosis. To determine if inhibition of these BCL-2 family members would further sensitize these MDOCS to copanlisib and TAK-228, these inhibitors were tested in combination with navitoclax (ABT-263; BCL-2 family inhibitor). A dramatic enhanced sensitivity was observed in MDOCS (30% p-value<0.001, 23% p-value<0.001, respectively). This correlated with an induction of apoptosis as measured by cleaved caspase 3. Next a panel of eight CRC patient-derived organotypic cancer spheroids (PDOCS) were treated with the combination of TAK-228 and navitoclax. Differential sensitivity was observed across the panel (25% resistant, 37.5% intermediate, and 37.5% highly sensitive) owing to the importance of mutational profile with targeted therapies. These studies indicate the benefit of MTORC1/2 for the treatment of PIK3CA mutant CRC and with enhanced activity of the combination of MTORC1/2 inhibition in combination with BCL-2 family inhibition. These therapies deserve further investigation for the treatment of patients with PIK3CA mutant CRC. Citation Format: Rebecca A. DeStefanis, Susan N. Payne, Devon Miller, Cheri A. Pasch, Christopher Babiarz, Alyssa DeZeeuw, Stephanie L. Fricke, Carley Sprackling, Alexander E. Yueh, Demetra P. Korkos, Dana R. Van De Hey, Gioia Sha, Aurora Greane, Jeremy D. Kratz, Linda Clipson, Kristina A. Matkowskyj, Michael A. Newton, Dustin A. Deming. MTORC1/2 inhibition as a treatment strategy for PIK3CA mutant colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B27.

Abstract Colorectal cancer (CRC) is a leading cause of cancer related death with PIK3CA mutations occurring in ~18% of all cases. Mutations in this gene lead to constitutive activation of the phosphoinositide-3 kinase (PI3K) oncogene. Previously we've shown that MTORC1/2 inhibition is sufficient to induce a therapeutic response both in vitro and in vivo with minimal induction of apoptosis. BCL-xL is a well-known negative regulator of apoptosis in solid tumors. We therefore investigated whether inhibition of the BCL-2 family, and more specifically BCL-xL, would enhance therapeutic response and induction of apoptosis. Murine-derived cancer organoids (MDCOs) were generated from invasive colon adenocarcinomas of Apc and Pik3ca transgenic mice (F1 (FVBxB6) Apcfl/+ Pik3caH1047R). MDCOs were allowed to mature for 24 hours, baseline brightfield imaging performed and therapeutic agents added at concentrations outlined below. Median relative change in organoid diameter after 48 hours of treatment was determined. In vivo response was measured in F1 (FVBxB6) Apcfl/+ Pik3caP110* mice as change in endoscopic tumor lumen occlusion over 14 days. Immunoblotting (IB) and immunofluorescence (IF) were utilized to evaluate for induction of apoptosis. Navitoclax (BCL-2/BCL-xL/BCL-w inhibitor, 250nM) was evaluated alone and in combination with a panel of MTORC1/2 inhibitors (BEZ-235 (BEZ), TAK-228 (TAK), copanlisib (Cop), 200nM). Navitoclax did not induce a treatment response as a single agent. Enhanced response was seen with the combination compared to the MTORC1/2 inhibitors alone (Bez 56% vs combo -100%, p<0.001; TAK -27% vs combo -100%, p<0.001; Cop -16% vs -100%; p<0.001). Results were confirmed in vivo with BEZ-235 (30mg/kg/day), navitoclax (80mg/kg/day), or the combination with the greatest reduction in lumen occlusion of colon tumors in the combination therapy (control +15%, navitoclax +1%, BEZ -15%, and combo -42%, p<0.003 BEZ vs combo). IB of cleaved PARP, a main cleavage target of cleaved caspase 3 (CC3) once apoptosis is induced, and IF of CC3 confirmed induction of apoptosis was highest in the combination therapy in both in vitro and in vivo studies. This induction was found as early as 6 hours post treatment in the MDCOs. To confirm inhibition of BCL-xL was the primary anti-apoptotic protein necessary for this induction of apoptosis, MDCOs were treated with copanlisib (200nM) alone or in combination with WEHI-539 (BCL-xL inhibitor, 250nM) or ABT-199 (BCL-2 inhibitor, 250nM). An enhanced sensitivity was observed when MTORC1/2 inhibition was combined with the inhibition of BCL-xL compared to BCL-2. These studies indicate that BCL-xL signaling reduces MTORC1/2 inhibitor response and targeting BCL-xL in combination with MTORC1/2 enhances both the treatment response and the induction of apoptosis in PIK3CA mutant CRC. Citation Format: Rebecca A. DeStefanis, Alyssa DeZeeuw, Gioia Sha, Susan N. Payne, Christopher P. Babiarz, Devon Miller, Demetra K. Korkos, Cheri A. Pasch, Linda Clipson, Kristina Matkowskyj, Dustin A. Deming. BCL-xL inhibition enhances therapeutic response of MTORC1/2 inhibition and induction of apoptosis in PIK3CA mutant colorectal 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 75.

The normal colon epithelium is transformed into its neoplastic counterpart through a series of genetic alterations in driver genes including activating mutations in PIK3CA . Treatment often involves surgery followed by 5‐fluorouracil (5‐FU) based therapy, which has limited efficiency and serious side effects. We sought to determine whether fisetin, a dietary flavonoid, alone or in combination with 5‐FU affected tumorigenesis in the mammalian intestine. We first determined the effect of fisetin, 5‐FU or their combination on PIK3CA ‐mutant and PIK3CA wild‐type colon cancer cells by assessing cell viability, colony formation, apoptosis and effects on PI3K/AKT/mTOR signaling. Treatment of PIK3CA ‐mutant cells with fisetin and 5‐FU reduced the expression of PI3K, phosphorylation of AKT, mTOR, its target proteins, constituents of mTOR signaling complex and this treatment increased the phosphorylation of AMPKα. We then determined whether fisetin and 5‐FU together or singly affected tumorigenesis in Apc Min /+ mice that also express constitutively active PI3K in the distal small intestine and colon. Tumor incidence was markedly lower in fisetin‐treated FC 1 3K 1 Apc Min/+ mice that also express constitutively active PI3K in distal small intestine and colon, as compared to control animals, indicating that fisetin is a strong preventive agent. In addition, the combination of fisetin and 5‐FU also reduced the total number of intestinal tumors. Fisetin could be used as a preventive agent plus an adjuvant with 5‐FU for the treatment of PIK3CA ‐mutant colorectal cancer.

Abstract Background: There are limited clinical tools for predicting the effectiveness of cancer therapies. We aim to prospectively predict patient treatment response using patient-derived organotypic cancer spheroids (PDOCS) as an in vitro model which recapitulates the genetic characteristics and 3D organization of the patient’s tumor. Using optical metabolic imaging (OMI) to analyze single cells, we can determine heterogeneous subpopulations in response to drug treatment. Further clinical validation of these techniques and analysis methods are needed before clinical translation. Methods: Tissue biopsies and gross tissue resections were acquired through the University of Wisconsin Precision Medicine Molecular Tumor Board (IRB#UW15068) and UWCCC TSB Biobank. Next-generation sequencing (NGS) from the biopsies was performed to determine molecular profiling. In alignment with the patient’s treatment course, PDOCS were treated with physiologic doses of chemotherapy or targeted therapy. Treatment response was evaluated by measuring change in diameter in conjunction with optical metabolic imaging (OMI) using a multiphoton microscope to measure the fluorescence and redox ratio of NAD(P)H and FAD as an indication of cellular metabolism. Diameter changes between control and treatment groups were compared using Glass’s delta; resistance to therapy was indicated by a Glass’s delta score of below 1.5. The optical redox ratios determined by OMI were compared using Glass’s delta, and resistance was indicated below 0.5. Clinical response was measured using RECIST v1.1 standard response assessment criteria. Results: PDOCS were successfully isolated from colorectal (CRC), lung, gastrointestinal stromal tumor (GIST), ovarian, and breast cancers. These biopsies were all obtained in the treatment refractory setting. PDOCS were generated for seven patients and treated with the same pharmacologic treatment as the patient from which the PDOCS were generated. Multiple treatments were able to be tested both in vitro and clinically for a subset of patients. Treatments included: 5-fluouracil, oxaliplatin, gemcitabine, paclitaxel, olaparib, panitumumab, osimertinib, fulvestrant, and palbociclib. In this cohort, two treatments resulted in stable disease and seven treatments resulted in disease progression. Change in spheroid diameter correlated with clinical treatment outcomes with an effect size (Glass’s delta) threshold of 1.5. OMI predicted response for all patients imaged with an effect size threshold of 0.5 which correlated with the size change analyses. Treatment heterogeneity of OMI was observed in many of the samples. Conclusions: In this largely prospective cohort of patients across disease types, changes in PDOCS size and OMI indices predict treatment benefit for individual patients. Studies on a larger scale are needed to further validate these findings. Citation Format: Carley M. Sprackling, Jeremy D. Kratz, Peter F. Favreau, Mohammad R. Karim, Christopher P. Babiarz, Cheri A. Pasch, Amani A. Gillette, Linda Clipson, Kristina A. Matkowskyj, Jens C. Eichoff, Kayla K. Lemmon, Hannah K. Houtler, Mark E. Burkard, Devon Miller, Melissa C. Skala, Dustin A. Deming. Predicting treatment response using patient derived organotypic cancer spheroids [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 3143.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the United States, despite substantial development in its early diagnosis and treatment. The animal models of CRC have been immensely useful for understanding CRC pathogenesis, investigating the effects of genetic modifications on CRC, and for the development of new chemopreventive/chemotherapeutic drugs. Most (>80%) CRC carry mutations in the APC gene and many (15-20%) carry mutations in the PIK3CA gene, encoding the p110 catalytic subunit of the PI3K kinase. We sought to better understand the interaction between APC and PIK3CA mutations in the mammalian intestine. To examine the effect of mutations in APC and PIK3CA on tumorigenesis, Min mice were crossed with the FC13K1 (FC13K1ApcMin/+). This cross resulted in a murine model with the loss of one allele of Apc throughout the intestine and the expression of a dominant active PI3K (3K1) in the distal small intestine and colon due to the expression of Cre under the control of the rat fatty acid binding protein-1 promoter (FC1). There was increased tumor multiplicity, size and a more aggressive and poorly differentiated phenotype as a consequence of synergy between APC and PIK3CA mutations. Tumors form as adenomas, but quickly progress to invasive adenocarcinomas that eventually metastasize to regional lymph nodes. Using this mouse model, we have recently shown that fisetin, a dietary flavonoid could be used as a preventive agent and an adjuvant with 5-fluorouracil (FU) for the treatment of PIK3CA-mutant CRC. Tumor incidence was markedly lower in fisetin-treated FC13K1ApcMin/+ mice in distal small intestine and colon, as compared to control animals, indicating that fisetin is a strong preventive agent. In addition, the combination of fisetin and 5-FU also reduced the total number of intestinal tumors. We extended our work by demonstrating that the effect of drugs on tumorigenesis was impacted by the mutation profile of the tumor and intratumoral heterogeneity. We developed the mouse model in which intestinal tumors were composed entirely of PIK3CA wild-type cells, entirely of PIK3CA-mutant cells, or a mixture of both. We demonstrated that low dose aspirin blocked the development of heterogeneous tumors composed of PIK3CA wild-type and PIK3CA-mutant cells but not the development of homogenous tumors composed entirely of PIK3CA wild-type cells. Thus, this new model of CRC recapitulates the effect of aspirin that was observed in humans. Sustained exposure to low dose aspirin reduced the recurrence the PIK3CA-mutant CRC cancers in humans. This aggressive murine model is an exciting model of human CRC that has the potential to be instrumental in the development of targeted chemoprevention and therapeutics. Citation Format: Naghma Khan, Farah Jajeh, Emily L. Eberhardt, Devon D. Miller, Dawn M. Albrecht, Rachel Van Doorn, Melissa D. Hruby, Morgan E. Maresh, Linda Clipson, Hasan Mukhtar, Richard B. Halberg. Novel mouse model carrying APC and PIK3CA mutations in colorectal cancer [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 2733.

Abstract Purpose: Cancer treatment is limited by inaccurate predictors of patient-specific therapeutic response. Therefore, some patients are exposed to unnecessary side effects and delays in starting effective therapy. A clinical tool that predicts treatment sensitivity for individual patients is needed. Experimental Design: Patient-derived cancer organoids were derived across multiple histologies. The histologic characteristics, mutation profile, clonal structure, and response to chemotherapy and radiation were assessed using bright-field and optical metabolic imaging on spheroid and single-cell levels, respectively. Results: We demonstrate that patient-derived cancer organoids represent the cancers from which they were derived, including key histologic and molecular features. These cultures were generated from numerous cancers, various biopsy sample types, and in different clinical settings. Next-generation sequencing reveals the presence of subclonal populations within the organoid cultures. These cultures allow for the detection of clonal heterogeneity with a greater sensitivity than bulk tumor sequencing. Optical metabolic imaging of these organoids provides cell-level quantification of treatment response and tumor heterogeneity allowing for resolution of therapeutic differences between patient samples. Using this technology, we prospectively predict treatment response for a patient with metastatic colorectal cancer. Conclusions: These studies add to the literature demonstrating feasibility to grow clinical patient-derived organotypic cultures for treatment effectiveness testing. Together, these culture methods and response assessment techniques hold great promise to predict treatment sensitivity for patients with cancer undergoing chemotherapy and/or radiation.

Abstract Background: Colorectal cancer (CRC) is a leading cause of cancer-related death in the United States. Approximately 20% of human CRCs possess mutations in the PIK3CA gene, resulting in a constitutively active form of phosphoinositide-3 kinase (PI3K). These PIK3CA mutations most commonly occur concomitantly with the loss of adenomatous polyposis coli (APC). Copanlisib is a beta-isoform sparing PI3K inhibitor. Here we examine the response of CRCs with Apc and Pik3ca mutations to copanlisib alone and in combination with navitoclax, a BCL-2, BCL-xL, and BCL-w inhibitor. Methods: Murine-derived organotypic cancer spheroids (MDOCS) with Apc and Pik3ca mutations generated from transgenic mice were cultured in Matrigel and treated by exchanging feeding media containing desired concentration of each agent over the spheroids. Spheroids were treated with feeding media (control), 200nM ABT263, 200nM copanlisib, or the combination of both treatments. The spheroid culture response was quantified as the median relative change in the sphere diameter, comparing pre- and posttreatment 4x optical microscopic images to those obtained 48 hours post-treatment. Results: Untreated MDOCS had a median growth in sphere diameter of 137%. ABT263 did not significantly affect growth; however, copanlisib significantly reduced median sphere size by 24.6% (p<0.001) and the combination treatment reduced median sphere size by 33% which was significant to both control and copanlisib only treated spheres (p<0.001 and p=0.014, respectively). Phosphorylation of ribosomal protein S6 and 4EBP1 were suppressed with copanlisib treatment. Induction of apoptosis in these spheres treated with the combination regimen was confirmed with immunofluorescence for cleaved caspase 3 with minimal apoptosis observed in those spheres treated with copanlisib alone. Conclusion: In this study, we demonstrated the ability of copanlisib alone and combined with ABT263 to cause a marked decrease in sphere size of CRC MDOCS with Apc and Pik3ca mutations. Future tests will examine this novel regimen in vivo and potentially in future clinical trials. Citation Format: Devon D. Miller, Christopher P. Babiarz, Susan N. Payne, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Dustin A. Deming. Dual PI3K/BCL-2 family inhibition in colorectal cancers with Apc and Pik3ca mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 304.

Abstract Colorectal cancer (CRC) is the third most common cause of cancer-related deaths in the United States and is one of the most frequent and deadliest cancers worldwide. The relative survival rate is very poor for patients with metastatic CRC, regardless of the progress in chemotherapy. 5-Fluorouracil (5-FU) is widely used chemotherapeutic drug for the treatment of colon cancer, and the efficacious tumor therapy is generally obstructed by the progression of tumor resistance mechanisms. PIK3CA is one of the most frequently mutated genes in CRC, as about 15-20% of advanced CRC harbor activating mutations in PIK3CA exon 9 and/or exon 20. Therefore, modeling the effects of this type of mutation in the mammalian colon is significant. Fisetin (3,3',4',7-tetrahydroxyflavone), a naturally occurring flavonoid, is present in several fruits and vegetables. We have earlier shown that treatment of PIK3CA-mutant colon cancer cells with fisetin and 5-FU caused induction of apoptosis, decrease in the expression of PI3K, phosphorylation of Akt, phosphorylation of mTOR and its target proteins with an increase in the phosphorylation of AMPKα. Based on these exciting results, we determined the effect of the treatment of combination of fisetin and 5-FU on the multiplicity of colonic tumors and on the regression of invasive adenocarcinomas in FC13K1ApcMin/+ mice. In this in-vivo model of colon cancer, synergy exists between the loss of the tumor suppressor APC and the presence of a dominant active PI3K. The tumors form as adenomas, progress to invasive adenocarcinomas and eventually metastasize to regional lymph nodes, as observed in human patients. The anti-tumorigenic properties of fisetin were tested by treating these mice either early when mice had no or few visible colon polyps and late when mice had developed cancers. The treatment regime was the same for both groups. The only difference was age when treatment initiated; the early group started treatment between 25 and 30 days, whereas late group started treatment between 45 and 50 days. In the early treatment groups, fewer fisetin-treated mice developed tumors than controls and fisetin-treated animals bearing tumors developed fewer colon tumors than controls. We also found that 5-FU suppressed growth of tumors and the effects of fisetin and 5-FU are additive. At the end of the experiment, as compared to control group, there was 27% decrease in tumor counts on treatment of animals with 5-FU, 36% decrease with fisetin and 49% decrease on treatment with combination of fisetin and 5-FU. There was also decrease in tumor incidence on treatment of animals with fisetin and combination of fisetin and 5-FU. Treatment with fisetin, 5-FU, or the combination did not appear to affect tumorigenesis when given late. Interestingly, the activation of PI3K appears to make 5-FU less effective at this stage. Our data suggest that fisetin could be used as a preventive agent as well as an adjuvant with 5-FU for the prevention of PIK3CA-mutant CRC. Citation Format: Naghma Khan, Farah Jajeh, Devon Miller, Rachel Van Doorn, Emily Lauren Eberhardt, Richard B. Halberg, Hasan Mukhtar. Fisetin for the management of PIK3CA-mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1251.

Abstract Colorectal cancer (CRC) is one of the most frequent and deadliest cancers worldwide with patients often diagnosed in advanced stages of the disease. Approximately 15-20% of advanced colorectal cancers harbor activating mutations in PIK3CA, which has been identified as an important oncogene in multiple cancers. Therefore, modeling the effects of this type of mutation in the mammalian colon is significant. Fisetin is a naturally occurring flavonoid in strawberry, apple, persimmon, grape, onion and cucumber. 5-Fluorouracil (5-FU) is the most used chemotherapeutic agent in CRC; however, it has serious side-effects. Therefore, augmentation of the 5-FU therapeutic effect could lead to lower effective doses and subsequently fewer side effects. We conducted in-vitro and in-vivo studies to determine the effect of fisetin, 5-FU and their combination on PI3K/AKT/mTOR signaling in PIK3CA-mutant colon cancer cells (HCT116 and HT-29), PIK3CA wild-type colon cancer cells (SW480), and newly developed mouse models. We found that there was more pronounced decrease in cell-viability and number of colonies in PIK3CA-mutant colon cancer cells than PIK3CA wild-type colon cancer cells. Apoptotic genes and proteins are promising targets for cancer treatment as they provide several theoretical basis to influence pathways causing greater tumor cell death. We observed an increase in the protein expression of Bax and decrease in Bcl2 on treatment with combination of fisetin and 5-FU than either agent alone. The full size PARP (116 KD) protein was also cleaved to yield an 85 KD fragment after treatment of cells with fisetin, 5-FU and their combination. Apoptotic effects of fisetin and 5-FU combination were also confirmed by flow cytometry in PIK3CA-mutant colon cancer cells. The PI3K/AKT pathway is frequently activated in CRC leading to tumorigenesis and the resistance to chemotherapy. Treatment of PIK3CA-mutant colon cancer cells with fisetin and 5-FU caused decrease in the expression of (i) PI3K (p85 and p110), (ii) phosphorylation of Akt (Ser473 and Thr308), (iii), phosphorylation of mTOR, its target proteins, and constituents of mTOR signaling complex. Treatment with fisetin and 5-FU also led to an increase in the phosphorylation of AMPKα. Next, we performed studies to investigate the effect of the treatment of combination of fisetin and 5-FU on colorectal tumorigenesis in FC13K1ApcMin/+ mice. These animals form tumors in the distal small intestine and colon that have lost APC activity and express constitutively active PI3K as often occurs in humans. Interestingly, the effect of fisetin was much stronger than that of 5-FU and comparable to the fisetin and 5-FU combination. Both fisetin only and combination treatment groups had significantly lower incidence relative to the control group. We suggest that fisetin could be used as a preventive agent as well as an adjuvant with 5-FU for the treatment of PIK3CA-mutant CRC. Citation Format: Naghma Khan, Farah Jajeh, Devon Miller, Rachel Van Doorn, Richard B. Halberg, Hasan Mukhtar. Fisetin, a dietary flavonoid for the prevention and treatment of PIK3CA-mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2030. doi:10.1158/1538-7445.AM2017-2030