PIK3CA Mutant Colorectal Cancer Research Articles

Abstract 355: BCLxL inhibition enhances copanlisib response in colorectal cancer

Abstract Background: Anti-apoptotic signaling, including through BCLxL, is a potential mechanism of resistance to PI3K inhibition in PIK3CA mutant cancers. We have previously demonstrated enhanced activity of PI3K inhibition in combination with BCLxL inhibition in PIK3CA mutated colorectal cancer (CRC) models. Here we examine the potential impact of BCLxL expression in PIK3CA mutant CRCs and the mechanism of treatment response with the combination of copanlisib and navitoclax. Methods: PIK3CA mutant CRCs were identified within TCGA colorectal adenocarcinoma data set and two cohorts were generated based on BCL2L1 (gene that encodes protein BCLxL) expression. Differentially expressed genes and gene set enrichment analysis were performed. CRC cell lines SW48 and SW48PK (Horizon Discovery, SW48 with a PIK3CAH1047R/+ mutation) were treated with copanlisib (copan) (1-100 nM) and navitoclax (250 nM). Immunoblotting was performed quantified using ImageJ to evaluate PI3K and apoptotic signaling. Flow cytometry using annexin V and propidium iodide (PI) was performed as a measure of apoptosis. Immunohistochemistry (IHC) using Ki67 was performed as a measure of cell proliferation on SW48PK xenografts and quantified as percent positive nuclei at 40x field of view (FOV). Results: Two datasets comprised of PIK3CA mutant CRC patients with either high or low BCL2L1 expression containing 39 and 47 patients, respectively, were used in the TGCA analysis. There was no difference in age, gender, or staging between these two datasets. An increase in DNA repair (false discovery rate q-value= 0.034) and interferon alpha signaling pathways (q= 0.022) was seen in PIK3CA mutant and high BCL2L1 expression compared to PIK3CA mutant and low BCL2L1 expression patients. PIK3CA mutant patients with low expression of BCL2L1 had a greater disease-free and overall survival (p= 0.037 and p= 0.042, respectively). Combination of copan and navitoclax treatment of the SW48 and SW48PK cell lines resulted in a reduction in activated ribosomal protein S6 (pRPS6) as compared with control or navitoclax alone at both 6 and 24. Cleaved PARP increased in SW48PK cells treated with 100 nM copan plus navitoclax versus control at both 6 and 24 hour timepoints (p= 0.03, p= 0.049). SW48 and SW48PK cells that were treated with copan and navitoclax for 24 hours had an increase in annexin v, a marker for apoptosis, compared to cells treated with control or single agents. In SW48PK xenografts, a significant reduction in Ki67 percent positive nuclei was seen in combination compared to control (median percent positive nuclei: 20.5, control: 33.2, p<0.001). Conclusions: High BCLxL expression is a sign of poor prognosis for PIK3CA mutant CRC. Inhibiting BCLxL can enhance the sensitivity of PI3K inhibition in PIK3CA mutant cancers and deserves further investigation clinically. Citation Format: Alexa E. Schmitz, Rebecca A. DeStefanis, Alyssa K. DeZeeuw, Autumn M. Olson, Anna L. Lippert, Kennedy J. Maduscha, Katherine A. Johnson, Sean Kraus, Cheri A. Pasch, Dustin A. Deming. BCLxL inhibition enhances copanlisib response in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 355.

Abstract 2965: GADD45B as a mechanism for enhanced treatment response with the combination of copanlisib and romidepsin in colorectal cancer

Abstract Background: Treatment options for molecular subtypes of colorectal cancer (CRC) are urgently needed, including for PIK3CA mutant CRC. Previous work in our lab demonstrated enhanced activity of copanlisib in combination with romidepsin in PIK3CA mutant CRC models. Here we investigate the induction of GADD45B as a mechanism of the enhanced induction of apoptosis seen with this treatment strategy. Methods: RNA sequencing was done on the CRC 2D cell line, SW48PK (Horizon Discovery, SW48 PIK3CAH1047R/+ mutation) as well as PIK3CA mutant and wild-type patient derived cancer organoids (PDCOs) through the UW Biotechnology Center. All lines were treated with copanlisib, romidepsin, or combination and were compared with untreated controls. GADD45B gene expression was also confirmed on SW48PK, PIK3CA mutant, wild type, and PIK3CA and KRAS mutant PDCOs using RT-qPCR. Results: RNA sequencing determined that there was an increase of GADD45B expression in the SW48PK 2D cell line as well as in PIK3CA mutant and wild-type PDCOs treated with the combination compared to untreated controls (average log2 fold change= 1.83). A significant increase in GADD45B expression was seen in the SW48PK combination treatment group compared to the control group at 6 hours (combination median fold change= 2.7, p= 0.045) and 24 hours (4.1, p< 0.001). For PIK3CA mutant PDCOs treated with the combination, there was significant increase in GADD45B expression at 6 hours (9.45, p= 0.005) and 24 hours (14.7, p= 0.002) compared to untreated control. In wild type PDCOs, there was a significant increase in GADD45B expression in the combination compared to untreated control at 6 hours (32.95, p=0.004) but not after 24 hours (2.6, p= 0.42). For KRAS mutant PDCOs, there was a significant increase in GADD45B expression in combination treated cells at 6 (9.9, p= 0.001) and 24 hours (3.3, p= 0.047), however, this increase was less than PIK3CA mutant PDCOs at 24 hours (3.3 vs 14.7). Conclusions: Copanlisib in combination with romidepsin increases GADD45B expression in PDCOs and SW48PK. Further studies will determine if GADD45B is required for this treatment response. Citation Format: Kennedy Maduscha, Alexa Schmitz, Rebecca DeStefanis, Cheri Pasch, Dustin Deming. GADD45B as a mechanism for enhanced treatment response with the combination of copanlisib and romidepsin in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2965.

Abstract 4125: Exploring the efficacy of alpelisib and combined PI3Kα-S6K inhibition in LIN28B-mediated colorectal cancer metastasis

Abstract Introduction: Colorectal cancer (CRC) is the third most diagnosed cancer worldwide. LIN28B, an RNA-binding protein that regulates mRNA translation, has garnered significant interest in recent cancer research. Our lab has established that LIN28B is an oncogene and promotes CRC metastasis. However, the exact mechanisms through which LIN28B exerts these effects remain elusive. The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is altered in 50-70% of CRCs and is characterized by PIK3CA gene mutations in 20-30% of CRC cases. PIK3CA encodes the catalytic subunit p110α of the PI3Kα enzyme, which is critical for PI3K/AKT signaling. Activation of this pathway is associated with worse CRC prognosis. Among the components of this signaling cascade, the ribosomal protein S6 kinase (S6K) is a crucial downstream effector, playing a pivotal role in protein synthesis and cell growth. Despite the frequent occurrence of PIK3CA mutations in CRC, targeted therapies for PIK3CA-mutant CRC are lacking. Alpelisib, a PI3Kα-specific inhibitor approved by the FDA for a subset of PIK3CA-mutant breast cancers, remains largely unassessed in CRC. We hypothesize that LIN28B-mediated CRC metastasis is facilitated by the activation of the PI3K/AKT pathway, representing a potential target for Alpelisib or other related inhibitors. Results: Our data indicate that LIN28B expression in CRC cells promotes liver metastasis in mouse models of metastatic CRC (mCRC). Furthermore, LIN28B expression activates the PI3K/AKT pathway, as measured by an AKT phosphorylation array. Treatment of CRC cells with SC79, a pan-AKT activator, enhanced cell migration and invasiveness. Similarly, VillinCre;R26Pik3ca transgenic mice, expressing a constantly active mutant form of PI3Kα in the intestinal epithelium, demonstrated crypt hyperplasia and an amplified propensity for tumorigenesis, mirroring the effects of LIN28B expression in CRC. Importantly, Alpelisib effectively hinders LIN28B-driven CRC progression in cell lines, transgenic mice-derived organoids, and mCRC mouse models, as well as the growth rate of CRC patient-derived organoids (PDOs). The effects observed with Alpelisib treatment were replicated with inhibition of S6K using either LY25284702 or PF4708671. Moreover, a combination of low-dose Alpelisib with low-dose LY25284702 or PF4708671 synergistically restricted the growth of PDOs. Conclusion: Our data provide compelling evidence that the PI3K/AKT pathway regulates LIN28B-mediated CRC metastasis, and its inhibition by Alpelisib is feasible. This underscores the potential for precision medicine in CRC, offering targeted therapies to PIK3CA-mutant CRC patients who lack specific treatment options. Furthermore, our study highlights the clinical advantages of combined inhibition strategies for improved anti-metastatic outcomes, a field in dire need of interventions in mCRC. Citation Format: Alice E. Shin, Kensuke Sugiura, Secunda W. Kariuki, David A. Cohen, Christopher J. Lengner, Peter A. Sims, Joel T. Gabre, Anil K. Rustgi. Exploring the efficacy of alpelisib and combined PI3Kα-S6K inhibition in LIN28B-mediated colorectal cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4125.

NETs unleashed: neutrophil extracellular traps boost chemotherapy against colorectal cancer.

Chemotherapy, which primarily acts on cancer cells, can influence the tumor microenvironment and the recruitment and behavior of stromal cells. In this issue of the JCI, Li et al. explored the potent anticancer effect of the combination of a glutaminase inhibitor (CB-839) and 5-FU against PIK3CA-mutant colorectal cancer tumors. This chemotherapy treatment strongly induced the recruitment of neutrophils that formed neutrophil extracellular traps in cancer, which actively killed cancer cells by inducing apoptosis. This study substantially advances our understanding of the multifaceted role of neutrophils and NETs in the outcome of anticancer treatment.

Neutrophil extracellular traps induced by chemotherapy inhibit tumor growth in murine models of colorectal cancer.

Neutrophil extracellular traps (NETs), a web-like structure of cytosolic and granule proteins assembled on decondensed chromatin, kill pathogens and cause tissue damage in diseases. Whether NETs can kill cancer cells is unexplored. Here, we report that a combination of glutaminase inhibitor CB-839 and 5-FU inhibited the growth of PIK3CA-mutant colorectal cancers (CRCs) in xenograft, syngeneic, and genetically engineered mouse models in part through NETs. Disruption of NETs by either DNase I treatment or depletion of neutrophils in CRCs attenuated the efficacy of the drug combination. Moreover, NETs were present in tumor biopsies from patients treated with the drug combination in a phase II clinical trial. Increased NET levels in tumors were associated with longer progression-free survival. Mechanistically, the drug combination induced the expression of IL-8 preferentially in PIK3CA-mutant CRCs to attract neutrophils into the tumors. Further, the drug combination increased the levels of ROS in neutrophils, thereby inducing NETs. Cathepsin G (CTSG), a serine protease localized in NETs, entered CRC cells through the RAGE cell surface protein. The internalized CTSG cleaved 14-3-3 proteins, released BAX, and triggered apoptosis in CRC cells. Thus, our studies illuminate a previously unrecognized mechanism by which chemotherapy-induced NETs kill cancer cells.

MO32-5 Metabolic interaction between gut microbiome and PIK3CA-mutated colorectal cancer: SCRUM-Japan MONSTAR-SCREEN

MO32-5 Metabolic interaction between gut microbiome and PIK3CA-mutated colorectal cancer: SCRUM-Japan MONSTAR-SCREEN

Aspirin promotes RSL3-induced ferroptosis by suppressing mTOR/SREBP-1/SCD1-mediated lipogenesis in PIK3CA-mutant colorectal cancer

Ferroptosis, a new form of regulated cell death triggered by the iron-dependent peroxidation of phospholipids, is associated with cellular metabolism, redox homeostasis, and various signaling pathways related to cancer. Aspirin is a widely used non-steroidal anti-inflammatory drug (NSAID) and has been reported to show therapeutic benefit in cancers harboring oncogenic PIK3CA, which encodes the catalytic p110α subunit of phosphoinositide 3-kinase (PI3K). In this study, we found that aspirin sensitized cancer cells harboring oncogenic activation of PIK3CA to ferroptosis induction. Mechanistically, aspirin inhibited protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling, suppressed downstream sterol regulatory element-binding protein 1 (SREBP-1) expression, and attenuated stearoyl-CoA desaturase-1 (SCD1)-mediated lipogenesis of monounsaturated fatty acids, thus promoting RSL3-induced ferroptosis in colorectal cancer (CRC) cells. Moreover, genetic ablation of SREBP-1 or SCD1 conferred cancer cells greater sensitivity to ferroptosis induction. Conversely, ectopic expression of SREBP-1 or SCD1 restored ferroptosis resistance in CRC cells and abolished the effect of aspirin on RSL3-induced cytotoxicity. Additionally, the synergistic effects of aspirin and RSL3 were confirmed in a xenograft mouse model. The combined use of aspirin and RSL3 resulted in significant tumor suppression. Our work demonstrated that aspirin enhanced the cytotoxic effect of RSL3 in PIK3CA-mutant cancers, and the combination of aspirin and ferroptosis inducer displayed promising therapeutic effects in cancer treatment.

Abstract 1128: MTORC1/2 and HDAC1/2 inhibition promote tumor response through inhibition of MYC

Abstract Background: The identification of treatment strategies targeting PIK3CA mutant colorectal cancer (CRC) are of great clinical interest. Previous work from our lab has identified MTORC1/2 and HDAC1/2 inhibition with copanlisib (cop; PI3K/MTOR) and romidepsin (romi; HDAC1/2) as a potential therapeutic strategy. We hypothesized that changes in c-MYC protein levels and c-MYC target gene (CTG) alterations might be a potential mechanism of action for this combination. Methods: Known CTGs were identified and their expression levels were examined in PIK3CA mutant vs WT CRCs using the cBioPortal Colorectal Adenocarcinoma (TCGA, PanCancer Atlas) dataset. Murine derived cancer organoids (MDCO) were generated from adenocarcinomas of Apc and Pik3ca mutant transgenic mice (F1 (FVB x B6) Apcfl/+ Pik3caH1047R/+). MDCO results were corroborated using the human 2D isogenic cell lines SW48 and SW48PIK3CA-H1047R (SW48PK) and RAS/RAF WT patient derived cancer organoids (PDCO) generated from CRC patient samples under approved IRB protocols. Immunoblots (IB) were used to assess c-MYC levels after treatment with cop, romi, and the combination across all models. RNA sequencing was conducted on PDCOs and SW48PK cells and changes in 16 CTGs were examined. An aggregate score was created for each treatment group where a statistically significantly altered CTG with log fold change ≥1.5 added one point and ≤-1.5 subtracted one point. All others were scored 0. Results: c-MYC target genes were assessed for differential expression in PIK3CA mutant CRC vs PIK3CA WT CRC with only 1/16 genes decreased in PIK3CA mutant CRC (GADD45A: log ratio -0.21, q=0.01). Next, MDCOs treated with the combination showed a decrease in total c-MYC levels in the combination therapy. These results were corroborated in two human 2D CRC cell lines, SW48 and SW48PK and a panel of PDCOs. Interestingly, c-MYC levels decreased in both romi alone and the combination in both the SW48 and SW48PK cell lines after 24 hours of treatment. However, the extent to which c-MYC levels were decreased was not as substantial in the panel of PDCOs. Across all in vitro models a decrease in PI3K signaling, as illustrated by decreased pRPS6 and p4EBP1 in response to cop treatment and an increase in H3K27 acetylation in response to romi, was observed in the single agent and combination therapies. RNA sequencing demonstrated that cop, romi, and combo had a CTG score of 0, -4, and -7 respectively in the PIK3CA mutant PDCO. Similar results were seen in an additional RAS/RAF WT PDCO (0, 1, and -4, respectively) and SW48PK cells (0, -3, and -7, respectively). Conclusion: A potential mechanism by which cop and romi treatment promote tumor response is through a decrease in c-MYC protein levels and expression of downstream c-MYC target genes. This regimen deserves further mechanistic investigations in vivo. Citation Format: Rebecca A. DeStefanis, Autumn M. Olson, Alyssa K. DeZeeuw, Susan N. Payne, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. MTORC1/2 and HDAC1/2 inhibition promote tumor response through inhibition of MYC [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 1128.

Abstract 3721: MTORC1/2 and HDAC1/2 inhibition as therapy for colorectal cancer with PIK3CA mutation

Abstract Introduction: Colorectal cancer (CRC) is a leading cause of cancer-related deaths. PIK3CA mutations are found in 18% of CRCs which lead to the constitutive activation of the PI3K/MTOR pathway and can promote tumorigenesis. Here, we examine the efficacy of the FDA approved inhibitors copanlisib, a PI3K/MTOR inhibitor, and romidepsin, a HDAC1/2 inhibitor, in CRC with a PIK3CA mutation. Methods: CRC mouse derived cancer organoids (MDCOs) were derived from Apc and Pik3ca mutant mice (Fc1Apcfl/+Pik3caH1047R/+). Brightfield images of the MDCOs were taken prior to treatment with copanlisib, romidepsin, or the combination and 48 hours post-treatment. The change in diameter of each organoid over the 48-hour treatment was determined. Additionally, immunoblotting was performed to confirm known targets of copanlisib and romidepsin were altered in response to drug treatment. MTORC1/2 and HDAC1/2 inhibition were also investigated in vivo. SW48 and SW48PIK3CA-H1047R (SW48PK) xenograft mice were treated with a vehicle control, copanlisib, romidepsin, or the combination therapy. Results: In the diameter analysis study, the combination therapy had the largest effect size compared to control (Glass’s Δ 2.82). Single agents copanlisib and romidepsin had smaller effect sizes (Glass’s Δ 1.75 and 2.04, respectively). Immunoblotting results indicated a decrease in phosphoAKT (Ser473) and pRPS6 (Ser235/236) in the copanlisib treated samples and an increase in H3K27 acetylation was seen in the romidepsin treated samples. There was also increased cleaved PARP, an indicator of apoptosis, in the romidepsin and combination therapy treatment groups. In vivo, SW48 xenografts showed a greater response in the combination therapy compared to either single agent therapy (median relative change in tumor volume: control=339%; combination therapy=107%(p-value<0.05), copanlisib=225%(p-value=0.17), romidepsin=200%). A similar trend was seen in the SW48PK xenograft mice (median relative change in tumor volume: control=241%; combination therapy=70% (p-value<0.05), copanlisib=132%(p-value<0.05), romidepsin=177%). Conclusion: MDCOs with Apc and Pik3ca mutations had an increased response to treatment with the combination therapy as compared to the single agents alone. Additionally, in vivo studies with human CRC xenografts showed enhanced inhibition of tumor growth with both MTORC1/2 and HDAC1/2 inhibition. These data demonstrate potential for this combination treatment strategy for the treatment of PIK3CA mutant CRC and this combination warrants further investigation in other models and clinically. Citation Format: Autumn M. Olson, Rebecca A. DeStefanis, Alyssa K. DeZeeuw, Susan N. Payne, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. MTORC1/2 and HDAC1/2 inhibition as therapy for colorectal cancer with PIK3CA mutation [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 3721.

PIK3CA mutations-mediated downregulation of circLHFPL2 inhibits colorectal cancer progression via upregulating PTEN

BackgroundPIK3CA mutation and PTEN suppression lead to tumorigenesis and drug resistance in colorectal cancer (CRC). There is no research on the role of circular RNAs (circRNAs) in regulating PIK3CA mutation and MEK inhibitor resistance in CRC.MethodsThe expression of circLHFPL2 in PIK3CA-mutant and wild-type cells and tissues was quantified by RNA-sequencing and qRT-PCR. CCK-8 assay and colony formation assay were used to evaluate cell viability. Annexin V/PI staining was implemented to assess cell apoptosis. Luciferase assay, biotin-coupled microRNA capture, and RIP assay were used to validate the interaction among potential targets. Western blotting and qRT-PCR assays were used to evaluate the expression of involved targets. Xenograft tumor in a nude mouse model was used to explore the role of circRNAs in vivo.ResultsRNA sequencing defined downregulated expression of circLHFPL2 in both PIK3CAH1047R (HCT116) and PIK3CAE545K (DLD1) cells. CircLHFPL2 was also downregulated in PIK3CA-mutant CRC primary cells and tissues, which was correlated with poor prognosis. CircLHFPL2 was mainly localized in the cytoplasm and its downregulation was attributed to the PI3K/AKT signaling pathway activated by phosphorylating Foxo3a. CircLHFPL2 inhibited PI3KCA-Mut CRC progression both in vitro and in vivo. Furthermore, our work indicated that circLHFPL2 acts as a ceRNA to sponge miR-556-5p and miR-1322 in CRC cells and in turn modulate the expression of PTEN. Importantly, circLHFPL2 was able to overcome PIK3CA-mediated MEK inhibitor resistance in CRC cells.ConclusionsDownregulation of circLHFPL2 sustains the activation of the PI3K/AKT signaling pathway via a positive feedback loop in PIK3CA-mutant CRC. In addition, downregulation of circLHFPL2 leads to MEK inhibitor resistance in CRC. Therefore, targeting circLHFPL2 could be an effective approach for the treatment of CRC patients harboring oncogenic PIK3CA mutations.

In silico approach to target PI3K/Akt/mTOR axis by selected Olea europaea phenols in PIK3CA mutant colorectal cancer

Worldwide disease burden of colorectal cancer (CRC) increasing alarmingly, but a suitable therapeutic strategy is not available yet. Abnormal activation of the PI3K/Akt/mTOR signalling because of mutation in the PIK3CA gene is a driving force behind CRC development. Therefore, this study aimed to comprehensively characterise the potential of phenolic compounds from Olea europaea against the PI3K/Akt/mTOR axis by using in silico methodologies. Molecular docking was utilised to study key interactions between phenolic compounds of O. europaea and target proteins PI3K, Akt, mTOR with reference to known inhibitor of target. Drug likeness and ADME/T properties of selected phenols were explored by online tools. Dynamic properties and binding free energy of target-ligand interactions were studied by molecular dynamic simulation and MM-PBSA method respectively. Molecular docking revealed apigenin, luteolin, pinoresinol, oleuropein, and oleuropein aglycone as the top five phenolic compounds which showed comparable/better binding affinity than the known inhibitor of the respective target protein. Drug likeness and ADME/T properties were employed to select the top three phenols namely, apigenin, luteolin, and pinoresinol which shown to bind stably to the catalytic cleft of target proteins as confirmed by molecular dynamics simulations. Therefore, Apigenin, luteolin, and pinoresinol have the potential to be used as the non-toxic alternative to synthetic chemical inhibitors generally used in CRC treatment as they can target PI3K/Akt/mTOR axis. Particularly, pinoresinol showed great potential as dual PI3K/mTOR inhibitor. However, this study needs to be complemented with future in vitro and in vivo studies to provide an alternative way of CRC treatment. Communicated by Ramaswamy H. Sarma

Abstract 1944: Navitoclax enhances the efficacy of copanlisib predominantly through inhibition of BCLxL in PIK3CA mutant colorectal cancer

Abstract Background: Mutations in PIK3CA occur in 18% of colorectal cancers (CRC) and result in constitutive activation of the PI3K pathway. Previous work from our lab has determined that MTORC1/2 inhibition, with copanlisib (cop), a PI3K/MTOR inhibitor, is sufficient to induce a treatment response. We hypothesized that the BCL2 family inhibitor navitoclax (nav), a BCL2/BCLxL inhibitor, would enhance the therapeutic response of cop and increase the induction of apoptosis. Methods: Murine derived cancer organoids (MDCOs) were generated from adenocarcinomas arising in Apc and Pik3ca mutant transgenic mice ((FVB x B6) F1 Apcfl/+ Pik3caH1047R/+). MDCOs were plated and matured for 24 hours. MDCOs were treated for 48 hours. Change in diameter was used to assess response. Additionally, human 2D isogenic cell lines SW48 and SW48PIK3CA-H1047R (SW48PK) were plated and treated for 48 hours. WST-1 proliferation assay was used to determine response. In vivo response was assessed as median relative change (MRC) in tumor volume of SW48 and SW48PK xenografts in immunocompromised Rag2-Il2rg-/- (R2G2) mice. Induction of apoptosis was determined with immunoblotting (IB) for PARP cleavage. Results: MDCOs were treated with cop (200nM) or nav (250nM) alone and in combination. Single agent cop resulted in -16% MRC (p<0.001). Nav alone did not have an effect (+95% p<0.07). Enhanced therapeutic response was seen in the combination therapy compared to cop alone (cop -16% vs combo -100%; p<0.001). In SW48 and SW48PK with cop (1nM or 10nM) or nav (250nM) alone and in combination, a greater response was seen in the combination therapy in both cell lines compared to either single agent alone (p<0.005). SW48 and SW48PK xenograft studies demonstrated significant activity of this combination in vivo. SW48 tumor growth was limited with either cop (10mg/kg), nav (80mg/kg), or the combination compared to control (control +5.0 MRC, cop +3.4, nav +2.6, combo +2.6; control vs combo p<0.01; cop vs combo p=0.29). Enhanced activity of this combination was observed in SW48PK tumors compared to SW48. Growth was significantly more limited with the combination (control +5.0 MRC, cop +3.4, nav +4.5, combo +0.7; control vs combo p<0.01; cop vs combo p=0.06). IB of cleaved PARP showed induction of apoptosis was the highest in the combination therapy in all in vitro models and seen as early as 6 hours post treatment. To identify which BCL2 family member nav was primarily targeting, cop was combined with WEHI539 (BCLxL inhibitor; 250nM) or ABT199 (BCL2 inhibitor; 250nM). BCL2 inhibition did not enhance the efficacy of cop in the MDCOs or isogenic lines. However, BCLxL inhibition did enhance the efficacy of cop in both in vitro models. Conclusion: These data indicate that navitoclax, through BCLxL inhibition, enhances the efficacy of copanlisib with a greater induction of apoptosis in several models of PIK3CA mutant CRC. Citation Format: Rebecca Anna DeStefanis, Alyssa DeZeeuw, Gioia Sha, Autumn Olson, Samantha J. Anderson, Christopher P. Babiarz, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. Navitoclax enhances the efficacy of copanlisib predominantly through inhibition of BCLxL in PIK3CA mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1944.

MO18-7 Combinatorial treatments of aspirin with glutaminolysis-targeting agents against PIK3CA-mutated colorectal cancer cells

MO18-7 Combinatorial treatments of aspirin with glutaminolysis-targeting agents against PIK3CA-mutated colorectal cancer cells

5-Fluorouracil Enhances the Antitumor Activity of the Glutaminase Inhibitor CB-839 against PIK3CA-Mutant Colorectal Cancers

PIK3CA encodes the p110α catalytic subunit of PI3K and is frequently mutated in human cancers, including ∼30% of colorectal cancer. Oncogenic mutations in PIK3CA render colorectal cancers more dependent on glutamine. Here we report that the glutaminase inhibitor CB-839 preferentially inhibits xenograft growth of PIK3CA-mutant, but not wild-type (WT), colorectal cancers. Moreover, the combination of CB-839 and 5-fluorouracil (5-FU) induces PIK3CA-mutant tumor regression in xenograft models. CB-839 treatment increased reactive oxygen species and caused nuclear translocation of Nrf2, which in turn upregulated mRNA expression of uridine phosphorylase 1 (UPP1). UPP1 facilitated the conversion of 5-FU to its active compound, thereby enhancing the inhibition of thymidylate synthase. Consistently, knockout of UPP1 abrogated the tumor inhibitory effect of combined CB-839 and 5-FU administration. A phase I clinical trial showed that the combination of CB-839 and capecitabine, a prodrug of 5-FU, was well tolerated at biologically-active doses. Although not designed to test efficacy, an exploratory analysis of the phase I data showed a trend that PIK3CA-mutant patients with colorectal cancer might derive greater benefit from this treatment strategy as compared with PIK3CA WT patients with colorectal cancer. These results effectively demonstrate that targeting glutamine metabolism may be an effective approach for treating patients with PIK3CA-mutant colorectal cancers and warrants further clinical evaluation. SIGNIFICANCE: Preclinical and clinical trial data suggest that the combination of CB-839 with capecitabine could serve as an effective treatment for PIK3CA-mutant colorectal cancers.

Abstract PR09: Targeting glutamine addiction of PIK3CA mutant colorectal cancers: From preclinical models to clinical trials

Abstract Glutamine addiction is a major metabolic reprogramming event that occurs in cancer cells. Many tumors exhibit oncogene-dependent addiction to glutamine. As a metabolite, glutamine is first converted to glutamate by glutaminase (GLS) and then to α-ketoglutarate (α-KG) by either a transaminase or a glutamate dehydrogenase. PIK3CA, which encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase α, is frequently mutated in human cancers, including 20-30% of colorectal cancer (CRC). We found that oncogenic PIK3CA mutations render CRCs addicted to glutamine through an upregulation of glutamate pyruvate transaminase 2 (GPT2), thereby facilitating conversion of glutamate to α-KG. Here, we report that CB-839, a potent GLS inhibitor developed by Calithera Biosciences, inhibits xenograft tumor growth of PIK3CA mutant, but not WT, colorectal cancers. Remarkably, combination of CB-839 with 5-fluorouracil (5-FU) induces regression of xenograft tumors in four different PIK3CA mutant CRC models. These exciting preclinical data prompted us to perform a phase I and phase II clinical trial to assess combination of CB-839 with capecitabine, an oral prodrug of 5-FU. For the phase I trial, patients with advanced solid tumors and disease progression on standard treatment, or for whom capecitabine is an acceptable treatment option, were enrolled. A standard 3+3 design was used to assess escalating doses of CB-839 (400-800 mg) and capecitabine (500-1000 mg/m2) in four dose levels. CB-839 was given orally twice daily continuously and capecitabine was given orally twice daily on days 14/21 of a 21-day treatment cycle. Dose-limiting toxicity period was 21 days. Restaging CT scans were obtained every 9 weeks with response assessment per RECIST. Adverse events were assessed per CTCAEv4. Sixteen patients were enrolled, with 7 patients being treated at the final dose level (CB-839 800 mg and capecitabine 1000 mg/m2). No dose-limiting toxicity was observed. Ten patients achieved stable disease as their best response with 9 being fluoropyrimidine resistant. Among the 16 patients, 12 were CRC patients who were 5-FU resistant. Consistent with our preclinical data, the median progress-free survival (PFS) of seven PIK3CA mutant CRC patients was 26 weeks, whereas the median PFS of five PIK3CA WT CRC patients was 16 weeks. Notably, the PFS of three PIK3CA mutant CRC patients was over six months (8, 9 and 13 months, respectively). A phase II trial of the drug combination in metastatic PIK3CA mutant CRC patients is ongoing. In summary, our preclinical and clinical studies suggest that targeting glutamine metabolism may be a promising approach to treat CRCs with PIK3CA mutations. This abstract is also being presented as Poster A06. Citation Format: Jennifer Eads, Yiqing Zhao, David Bajor, Zhenghe John Wang. Targeting glutamine addiction of PIK3CA mutant colorectal cancers: From preclinical models to clinical trials [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 PR09.

Abstract B27: MTORC1/2 inhibition as a treatment strategy for PIK3CA mutant colorectal cancer

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 75: BCL-xL inhibition enhances therapeutic response of MTORC1/2 inhibition and induction of apoptosis in PIK3CA mutant colorectal cancer

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.

Abstract 3048: Murine-derived colorectal cancer organoid culture high-throughput drug screening to identify novel combinations targeting PIK3CA mutant cancers

Abstract Introduction: Colorectal cancer (CRC) treatment is becoming increasingly individualized as clinical mutation profiling is a standard of care. Oncogenic mutations of the PIK3CA gene induce a constitutively active phosphatidylinositol 3-kinase with downstream effects that promote tumor initiation and progression. Effective clinical therapeutic strategies targeting this subtype of colorectal cancer have remained elusive. Here we develop a high-throughput organoid drug screening platform to examine combination strategies targeting PIK3CA mutant CRC. Methods: Murine-derived CRC organoids (MDCOs) were derived from a transgenic mouse model of PIK3CA and APC mutated colorectal cancer (F1 (FVBxB6) Apcfl/+ Pik3caH1047R), matured for 48 hours and plated in 96 well plates. A total of 96 compounds chosen from clinically available anticancer drugs and select drugs in clinical development were analyzed for their efficacy in reducing organoid growth on their own, as well as in combination with copanlisib (PI3K/MTOR inhibitor) over 48 hours. Select treatments were confirmed using our standard 24 well plate assay. Additionally, irinotecan (SN38; topoisomerase-I inhibitor), panitumumab (EGFR inhibitor), and olaparib (PARP inhibitor) were investigated alone and in combination with copanlisib based on clinical use for this population or recent promising clinical trial data. Results: SN38, olaparib, and panitumumab yielded insignificant changes in spheroid size in the combination regimen compared to copanlisib alone (median relative change in organoid diameter - copanlisib -8%, p<0.001; SN38 combo -8%, p=0.5; olaparib combo -13%, p=0.6; and panitumumab combo 1%, p=0.9). The MDCOs were successfully utilized for the high-throughput drug screen. Differential sensitivity was identified using change in organoid diameter without the need for reagents or dyes which could complicate the screening method. Of the 96 compounds investigated in the high-throughput screen, 12 resulted in enhanced treatment effect when combined with copanlisib. Three of these 12 compounds inhibit microtubule formation, and others inhibit components of the RAS pathway. Two of these drug combinations were examined further and significant treatment responses were confirmed. Conclusion: Advances in the understanding of the molecular basis of cancer allow for a more precise approach to cancer treatment. High-throughput techniques are needed to better identify novel treatment strategies for molecular subtypes. Here we describe a label free murine-derived cancer organoid method to screen drugs for combination treatment strategies. Citation Format: Alyssa K. DeZeeuw, Rebecca A. DeStefanis, Gioia Sha, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. Murine-derived colorectal cancer organoid culture high-throughput drug screening to identify novel combinations targeting PIK3CA mutant cancers [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 3048.

Deactivation of Glutaminolysis Sensitizes PIK3CA-Mutated Colorectal Cancer Cells to Aspirin-Induced Growth Inhibition.

Aspirin is one of the most promising over-the-counter drugs to repurpose for cancer treatment. In particular, aspirin has been reported to be effective against PIK3CA-mutated colorectal cancer (CRC); however, little information is available on how the PIK3CA gene status affects its efficacy. We found that the growth inhibitory effects of aspirin were impaired upon glutamine deprivation in PIK3CA-mutated CRC cells. Notably, glutamine dependency of aspirin-mediated growth inhibition was observed in PIK3CA-mutated cells but not PIK3CA wild type cells. Mechanistically, aspirin induced G1 arrest in PIK3CA-mutated CRC cells and inhibited the mTOR pathway, inducing the same phenotypes as glutamine deprivation. Moreover, our study including bioinformatic approaches revealed that aspirin increased the expression levels of glutaminolysis-related genes with upregulation of activating transcription factor 4 (ATF4) in PIK3CA-mutated CRC cells. Lastly, the agents targeting glutaminolysis demonstrated significant combined effects with aspirin on PIK3CA-mutated CRC cells. Thus, these findings not only suggest the correlation among aspirin efficacy, PIK3CA mutation and glutamine metabolism, but also the rational combinatorial treatments of aspirin with glutaminolysis-targeting agents against PIK3CA-mutated CRC.

Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer.

Activation of the epithelial-to-mesenchymal transition (EMT) program is a critical mechanism for initiating cancer progression and migration. Colorectal cancers contain many genetic and epigenetic alterations that can contribute to EMT. Mutations activating the PI3K/AKT signaling pathway are observed in >40% of patients with colorectal cancer contributing to increased invasion and metastasis. Little is known about how oncogenic signaling pathways such as PI3K/AKT synergize with chromatin modifiers to activate the EMT program. Lysine-specific demethylase 1 (LSD1) is a chromatin-modifying enzyme that is overexpressed in colorectal cancer and enhances cell migration. In this study, we determine that LSD1 expression is significantly elevated in patients with colorectal cancer with mutation of the catalytic subunit of PI3K, PIK3CA, compared with patients with colorectal cancer with WT PIK3CA. LSD1 enhances activation of the AKT kinase in colorectal cancer cells through a noncatalytic mechanism, acting as a scaffolding protein for the transcription-repressing CoREST complex. In addition, growth of PIK3CA-mutant colorectal cancer cells is uniquely dependent on LSD1. Knockdown or CRISPR knockout of LSD1 blocks AKT-mediated stabilization of the EMT-promoting transcription factor Snail and effectively blocks AKT-mediated EMT and migration. Overall, we uniquely demonstrate that LSD1 mediates AKT activation in response to growth factors and oxidative stress, and LSD1-regulated AKT activity promotes EMT-like characteristics in a subset of PIK3CA-mutant cells. IMPLICATIONS: Our data support the hypothesis that inhibitors targeting the CoREST complex may be clinically effective in patients with colorectal cancer harboring PIK3CA mutations.