Combination Of Copanlisib Research Articles

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 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.

Preliminary Analysis of an Ongoing Phase II Study of Response-Adapted Therapy with Copanlisib and Rituximab for Untreated Follicular Lymphoma

Introduction Frontline therapy for pts with follicular lymphoma (FL) is not based on the molecular profile of the tumor. The PI3K pathway is central to FL biology, but the molecular profile of tumors most sensitive to PI3K inhibition (PI3Ki) is unknown. Copanlisib inhibits both PI3Kα and δ isoforms, and we hypothesized that most pts with treatment naïve FL will have PI3Ki-sensitive tumors and durable complete responses (CRs) may be achievable with short durations of targeted therapy. A gene expression profiling (GEP) predictor utilizes a combination of the expression of 45 selected informative genes and an additional 19 housekeeping genes to identify FL pts at high-risk for early progression within 24 months (Wright et al. ASH Annual Meeting2022). We also hypothesized that copanlisib would induce durable CRs in high-risk pts by the GEP assay. Here, we report a preliminary analysis of an ongoing “window of opportunity” study of copanlisib followed by response-adapted copanlisib and rituximab in treatment-naïve pts with FL (NCT03789240). Methods Pts with untreated grade 1-2, 3A FL, ≥stage 2 are eligible if systemic therapy is indicated: symptoms, progressive lymphadenopathy, or organ compromise. Prior radiation is permitted. Eligibility includes age ≥18 and adequate organ function unless due to lymphoma. HIV, CMV, Hep B or C, and autoimmune conditions are excluded. Pts first receive copanlisib 60mg on days 1, 8, and 15 of a 28-day “window of opportunity” to explore the activity of copanlisib. Following the window, pts receive 6 cycles of copanlisib 60mg on days 1, 8, and 15 of a 28-day cycle along with rituximab 375mg weekly x 4 then on day 1 of each cycle and response is assessed with FDG-PET and CT scans. Pts without response after C6 are taken off study and those with a partial response (PR) can continue for an additional 6 cycles and then stop therapy. Pts who achieve a CR after 6 cycles stop therapy. No maintenance is given. Supportive care includesPCP prophylaxis. The primary endpoint is the CR rate with secondary endpoints of safety, duration of CR, and PFS. Exploratory objectives include identification of a predictive signature of PI3Ki response and the response rate in high-risk pts identified by the GEP assay. Results Twenty-eight pts have enrolled. Median age was 56y (range, 24-80). Seventeen (61%) and 12 (43%) pts had high-risk FLIPI and FLIPI-2 scores ≥3, respectively; 6 (21%) pts had Grade 3A FL, and 17 (61%) pts met two or more GELF criteria. After copanlisib monotherapy in the window, 27 (96%) pts had tumor reductions by CT with a median change of -38% (-62% to +16%) (Figure 1). Among 22 pts who completed 6 cycles, 21 (96%) responded, including 9 (41%) pts with PR and 12 (55%) pts with CR. Of 9 pts with PR who received an additional 6 cycles of therapy, only 1 (11%) converted to a CR bringing the overall CR rate to 59% (13/22 pts). After a median follow-up of 25.8 months, the 2-year PFS was 43.2% (95% CI, 20.7-64.0%). The median duration of CR was 29.7 months (95% CI, 6.3 months - not estimable) with 8 (62%) CRs ongoing (Figure 2). Toxicity was evaluated in 28 pts across 220 cycles. Most common were rash (64%), mucositis (50%), and nausea/vomiting (50%), all G1-2 except 1 (4%) pt with G3 mucositis. The most common ≥G3 toxicities were neutropenia and infections in 5 (18%) pts each. ≥G3 neutropenia occurred in 10 (5%) cycles, responded to therapy interruption and/or growth factors, and did not require dose reduction. Two (8%) pts had copanlisib dose reduced, 1 (4%) each due to rash and mucositis. Therapy was prematurely discontinued in 5 pts: 3 due to COVID-19, 1 for hepatotoxicity that resolved, and 1 due to infusion reaction to rituximab. Nine of 24 (38%) pts were poor risk by the GEP assay. Complete responses were observed in 71%, 67%, and 57% of pts categorized as high-risk by FLIPI, FLIPI-2, and GEP assay, respectively. The CR rate was not affected by bulky disease (any LN >7cm; p=1.00), high-risk FLIPI (p=0.19), high-risk FLIPI-2 (p=0.67), poor-risk GEP (p=1.00), or metabolic tumor volume (p=0.33). Conclusion Nearly all pts with treatment naïve FL have PI3Ki-responsive tumors, and the combination of copanlisib with rituximab achieved a CR rate of 59% at the end of induction therapy. The median duration of CR is nearly 30 months, including ongoing remissions without maintenance. CRs were observed in pts deemed high-risk by the GEP assay and FLIPI score. The safety profile is excellent, with mostly G1-2 toxicities manageable with supportive care.

Pan-PI3K inhibition with copanlisib overcomes Treg- and M2-TAM-mediated immune suppression and promotes anti-tumor immune responses

The PI3K pathway is one of the most frequently altered signaling pathways in human cancer. In addition to its function in cancer cells, PI3K plays a complex role in modulating anti-tumor immune responses upon immune checkpoint inhibition (ICI). Here, we evaluated the effects of the pan-Class I PI3K inhibitor copanlisib on different immune cell types in vitro and on tumor growth and immune cell infiltration in syngeneic murine cancer models. Intermittent treatment with copanlisib resulted in a strong in vivo anti-tumor efficacy, increased tumor infiltration of activated T cells and macrophages, and increased CD8+ T cell/regulatory T cell and M1/M2 macrophage ratios. The strong in vivo efficacy was at least partially due to immunomodulatory activity of copanlisib, as in vitro these murine cancer cells were resistant to PI3K inhibition. Furthermore, the combination of copanlisib with the ICI antibody anti-PD-1 demonstrated enhanced anti-tumor efficacy in both ICI-sensitive and insensitive syngeneic mouse tumor models. Importantly, in an ICI-sensitive model, combination therapy resulted in complete remission and prevention of tumor recurrence. Thus, the combination of ICIs with PI3K inhibition by intermittently dosed copanlisib represents a promising new strategy to increase sensitivity to ICI therapies and to treat human solid cancers.

Abstract 4861: Copanlisib enhances the effectiveness of anti-PD1 therapies for colorectal cancer

Abstract Introduction: Immunotherapies are increasingly being used for patients diagnosed with cancer, however, for metastatic colorectal cancer (CRC), more than 95% of patients have shown little to no clinical benefit to immunotherapy. Previous work from our lab has shown that copanlisib, a PI3K inhibitor, was found to enhance MHC class I expression in Kras mutant CT26 murine colon cancer cells. Here we examine the potential for anti-cancer activity with the combination of copanlisib and anti-PD1 treatments using this model. Methods: The impact of copanlisib on CT26 cells was performed using the WST assay and the expression of MHC class I was assessed using flow cytometry. CT26 flank allografts were generated in Balb/C mice and subsequently treated for 15 days with copanlisib (10 mg/kg), anti-PD1 (0.2 mg, BioXCell) or the combination. An IgG2a antibody (BioXCell) was used as a control. Tumors were measured twice a week using a caliper. Tumors were excised, and prepared for immunohistochemistry for CD8, CD4, perforin, granzyme B, and F480 was performed. Staining was quantified as the number of positive staining cells per 20X field of view (FOV). Results: CT26 viability in response to 200 nM copanlisib was relatively unchanged as compared with untreated controls. Via flow cytometry, a 75% increase in mean fluorescent intensity (MFI) for MHC class I was observed comparing control to copanlisib (p=0.003). In vivo, after 15 days there was no difference in the growth rate of those cancers treated with control versus copanlisib or anti-PD1. A significant reduction in growth rate was observed with the combination of anti-PD1 with copanlisib compared to the other arms (median fold change=3.62; control: 7.49, p=0.002, copanlisib: 8.88, p=0.003; anti-PD1: 8.93, p=0.002). There were no differences in CD8 T cell and perforin expression between the treatment groups. Granzyme B expression was higher in the combination compared (median/FOV= 44) to the control (27, p= 0.01). Additionally, a significant reduction in F480 expression was seen in the combination compared to the other treatment groups (median/FOV=43; control: 139, p<0.001, copanlisib: 110, p<0.001; anti-PD1:149, p<0.001). Conclusions: In conclusion, copanlisib in combination with anti-PD1 demonstrated enhanced anti-tumor activity in Balb/c mice that were injected with CT26 CRC cells. This response was correlated with increased granzyme B expression and a reduction in macrophages with the combination treatment. Further studies will expand on the mechanism of this combination therapy. Citation Format: Alexa E. Schmitz, Kennedy J. Maduscha, Sarbjeet Makkar, Cheri A. Pasch, Rebecca A. DeStefanis, Philip B. Emmerich, Dustin A. Deming. Copanlisib enhances the effectiveness of anti-PD1 therapies for colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4861.

PB2132: COPANLISIB PLUS RITUXIMAB-BENDAMUSTINE FOR RELAPSED-REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA: RECRUITMENT UPDATE ON AN ONGOING PHASE II TRIAL OF THE FONDAZIONE ITALIANA LINFOMI (FIL_COPA-RB)

Background: Diffuse large B cell lymphoma (DLBCL) is curable in 50-60% of cases with frontline RCHOP. Relapsed/refractory (R/R) DLBCL can be cured with salvage treatments in roughly 40-50% of cases with standard available treatments which include high dose-chemotherapy plus autologous stem cell transplant (ASCT) in fit patients, and chimeric antigen receptor T-cells (CAR-T), permitted as third or subsequent line in patients with no major comorbidities. The outcome of R/R DLBCL unresponsive or ineligible to ASCT or CAR-T is highly unfavorable and new treatment options are needed. Rituximab plus bendamustine (RB) is active in R/R DLBCL with overall response rates (ORR) of 40-60% but generally short-lasting. Copanlisib is an intravenous pan-class I PI3K inhibitor with predominant activity against PI3K-α and -δ isoforms, active as single agent in DLBCL with ORR 25% (NCT02391116). The combination of copanlisib plus RB (Copa-RB) has showed a safe toxicity profile in R/R indolent lymphomas (iNHL) (NCT02626455). Aims: To assess the safety and efficacy of Copa-RB followed by copanlisib maintenance in R/R DLBCL not eligible or relapsed after ASCT or CAR-T. Methods: FIL Copa-RB is a multicentric open-label single arm, phase II trial. An estimated 81 patients will be enrolled, from 30 centers in Italy. Main inclusion criteria are patients aged ≥ 18 affected by DLBCL (including de-novo DLBCL, DLBCL transformed by iNHL and high-grade lymphoma) or FL grade IIIb R/R after ≤ 3 previous lines of therapy, ineligible or R/R to ASCT or CART, ECOG PS ≤ 2. Patients with primary mediastinal B cell lymphoma histology, significant organ dysfunction, uncontrolled hypertension and hyperglycemia (HbA1c> 8.5%) are excluded. Treatment consists of an induction phase with six 28-day cycles of Copa-RB (copanlisib 60 mg intravenously on day (D) 1, D8 and D15, rituximab 375 mg/m2 on D1, bendamustine 90 mg/m2 on D1-D2) followed by a maintenance phase of twelve 28-day cycles of copanlisib 60 mg given on D1 and D15. The primary endpoint is progression free survival (PFS) defined as the time between the date of enrolment and the date of disease progression, relapse or death from any cause, whichever occurs earlier, with an expected improvement of 12-month PFS from 20% to 35%. Secondary endpoints include overall survival, ORR, complete response (CR) rate, duration of response and rate of response improvement during maintenance. Exploratory analysis will include association between outcome and cell of origin, MYC, BCL2 and BCL6 overexpression and rearrangements, and mutational analysis of selected genes possibly involved in predicting response to copanlisib. The expected study duration from first patient’s first visit to last patient’s last visit is approximately 4 years. Results: The recruitment started in November 2020; at March 1st, 27/81 patients have been enrolled. Results for this study are not yet available. Summary/Conclusion: The treatment of R/R DLBCL ineligible or R/R to ASCT or CAR-T is still an unmet clinical need and new therapeutic strategies are warranted. Copanlisib as single agent has been already shown a moderate activity in DLBCL and the association Copa-RB plus copanlisib maintenance could be a new treatment option to improve the outcome of these patients. Biomarkers studies may further elucidate patients who are more likely to respond to this treatment. Funding: The study is sponsored by the Fondazione Italiana Linfomi; Bayer partly supported the study and kindly provided Copanlisib; Sandoz kindly provided Rituximab.

Targeting PI3Kα/δ and the ErbB Family of Protein-Tyrosine Kinases in Cisplatin-Resistant Head and Neck Squamous Cell Carcinomas

<h3>Purpose/Objective(s)</h3> The mortality rate for advanced head and neck squamous cell carcinoma (HNSCC) remains very high due to cancer recurrence and metastasis. Cisplatin is a commonly used anti-cancer drug for treatment of recurrent and metastatic HNSCC, but the majority of patients will eventually develop resistance. Therefore, there is an urgent, yet still unmet, need for improved therapies for cisplatin-resistant HNSCC. Increasing evidence has demonstrated that ErbB family kinase and PI3K/Akt are the most important regulators and targets in HNSCC. Our goal is to find new therapeutics to treat cisplatin resistant HNSCC without use of cisplatin. Here, we determined the efficacy of a combination of the PI3Kinase inhibitor, Copanlisib with the ErbB inhibitor, Afatinib, on suppressing cisplatin resistant HNSCC <i>in vitro</i> and <i>in vivo</i>. <h3>Materials/Methods</h3> Both cisplatin sensitive (Cal27 and FaDu) and resistant (Cal27CP and FaDu-CP) HNSCC cells were treated with copanlisib, afatinib, or a combination of copanlisib and afatinib. The effects on cell signaling pathways were tested by Western blot assay and RT-PCR. Cell proliferation was determined by MTT assay. Apoptosis was assayed by Annexin V. Migration and invasion was monitored via the xCELLigence real-time cell system tic cells. Xenograft tumor formation was observed in nude mice. <h3>Results</h3> We found that the combination of Copanlisib and Afatinib completely blocked PI3K/Akt/mTOR pathways and ErbB kinase family and significantly suppressed cell proliferation, survival, migration, and invasion <i>in vitro</i>, as well as xenograft tumor formation in animals. <h3>Conclusion</h3> We found that the combination of Copanlisib and Afatinib completely blocked PI3K/Akt/mTOR pathways and ErbB kinase family and significantly suppressed cell proliferation, survival, migration, and invasion <i>in vitro</i>, as well as xenograft tumor formation in animals.

Abstract P5-16-08: Phase Ib/II trial evaluating safety and efficacy of copanlisib (PI3K inhibitor) and trastuzumab in pre-treated advanced HER2-positive breast cancer: Results from the PantHER study

Abstract Background: De novo and acquired resistance to HER2 directed therapy is frequently encountered. Upregulation of the phosphatidylinositol-3-kinase (PI3K) pathway is an important mediator of treatment resistance. This can occur through an activating mutation of the PIK3CA gene or PTEN loss. PIK3CA mutations are present in approximately 20% of HER2 positive breast cancers and as such, the PI3K pathway has emerged as an attractive target for restoring sensitivity to HER2 directed therapy. Methods: We performed a single arm, multicentre, open label Phase Ib/II trial. Patients (pts) with advanced HER2-positive breast cancer whose disease had progressed on at least 1 line of Trastuzumab/T-DM1 based treatment in the metastatic setting were eligible if they met following criteria: ECOG PS ≤ 2 and adequate organ function. Pts with treated, controlled brain metastases were permitted to enrol. Exclusion criteria included uncontrolled hypertension or diabetes mellitus. Pts on Phase IB were treated according to a 6+6 study design with a dose escalation schedule of Copanlisib IV (level 1 = 45mg, level 2 = 60mg) on Day 1, 8 and 15 of a 28-day cycle along with a fixed dose of Trastuzumab 2mg/kg weekly. Phase II treatment was the MTD (maximum tolerated dose) of Copanlisib in combination with Trastuzumab. Archival tumour tissue, voluntary biopsies and serial plasma samples were collected for genomic sequencing. Primary endpoints were MTD (Phase I) and clinical benefit rate (CBR) which was defined as complete response (CR) or partial response (PR) at any time point; or stable disease (SD) lasting at least 24 weeks (Phase II). Secondary endpoints included safety and tolerability, tumor response rate, duration of response, time to treatment failure (TTF) and progression free (PFS) and overall survival (OS). Results: Twelve pts were enrolled in Phase IB. No dose limiting toxicity was observed. The MTD was established as Copanlisib 60mg and Trastuzumab 2mg/kg. Fourteen pts were enrolled in Phase II (6 pts treated at the MTD in Phase IB were included in the final Phase II analysis resulting in a total of 20 pts). The median number of lines of prior treatment in the metastatic setting was 3 (1-8). The most common grade 3-4 toxicities encountered in the Phase Ib/II cohorts included hypertension (n=7, 27%), hyperglycaemia (n=2, 8%) and vomiting (n=2, 8%). Three pts discontinued treatment due to toxicity. The median follow-up for the Phase II cohort was 7.5 months (95% CI 6.0-14.5). PR was observed in 4 pts (20%) and SD (at any time point) was seen in 8 pts (40%). The CBR was 30% (n=6). The duration of response was 15.0 weeks (95% CI 4.9 - 16.1). The median TTF was 11.9 weeks (95% CI 7.5 - 21.1). The median PFS was 3.0 mo (95% CI 0.2 - 5.8) and OS was 14.0 mo (95% CI 5.2-22.8). At the time of analysis, 9 of 20 patients were alive. PIK3CA mutations were detectable in the archival tissue of 11 of 26 pts (42%). PIK3CA hotspot mutations (H1047R, E542K and E545K) were detectable in the plasma of all 26 pts at various points throughout treatment. Pre and post treatment biopsies of 2 pts in the Phase IB trial revealed somatic mutations in DNAH3 and TRRAP, the latter of which encodes a PI3K-like protein kinase. Targeted next generation sequencing was performed on the circulating tumour DNA of 20 pts in the Phase II cohort taken before, during and after treatment to further validate these findings and to assess for other mechanisms of response or resistance. The final translational results will be presented at the meeting. Conclusions: The combination of Copanlisib and Trastuzumab is a safe and tolerable regimen and is associated with clinical efficacy in a heavily pre-treated metastatic HER2-positive breast cancer population. Translational studies may have identified novel resistance biomarkers in this pt cohort. Citation Format: Lisa Prior, Niamh M Keegan, Simon J Furney, Janice M Walshe, Giuseppe Gullo, John Crown, M John Kennedy, Diarmuid Smith, John McCaffrey, Catherine M Kelly, Keith Egan, Jennifer Kerr, Mark Given, Niall Sheehy, Peter O'Donovan, Andres Hernando, Ausra Teiserskiene, Imelda Parker, Elaine Kay, Ray McDermott, Maccon M Keane, Seamus O'Reilly, Liam Grogan, Oscar Breathnach, Patrick G Morris, Sinead Toomey, Bryan T Hennessy. Phase Ib/II trial evaluating safety and efficacy of copanlisib (PI3K inhibitor) and trastuzumab in pre-treated advanced HER2-positive breast cancer: Results from the PantHER study [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 P5-16-08.

Abstract CT222: BrUOG360: A phase Ib/II study of copanlisib combined with rucaparib in patients with metastatic castration-resistant prostate cancer (mCRPC)

Abstract Background: Patients with metastatic prostate cancer invariably progress to a lethal state called metastatic castration-resistant prostate cancer (mCRPC). Approximately 23-28% of patients with mCRPC have molecular alterations in genes associated with homologous recombination (HR) DNA repair mechanisms (e.g., BRCA1/2). These alterations are associated with sensitivity to poly ADP-ribose polymerase inhibitors (PARPi). The responses to PARPi can be short-lived, and several mechanisms of resistance have been described. Preclinical studies demonstrated that PI3K suppression impairs HR and sensitizes cancer cells to PARPi even in the absence of mutations in DNA repair genes. The synergy between olaparib and PI3K inhibition led to marked tumor regression both in vitro and in vivo models of prostate cancer. These preclinical results, coupled with the importance of both the PI3K pathway and DNA damage repair mechanisms in mCRPC, support our hypothesis that dual inhibition of PI3K and PARP might improve clinical outcomes in mCRPC. We describe an ongoing phase Ib/II study aimed to evaluate the safety and determine the maximum tolerated dose (MTD) of the combination of copanlisib (pan-class I PI3K inhibitor) and rucaparib (a PARP-1, -2 and -3 inhibitor) followed by an expansion cohort exploring efficacy among patients with mCRPC. Methods: Phase I, dose-escalation part of the study, aims to establish the MTD and/or recommended phase II dose (RP2D) of copanlisib in combination with rucaparib. Dose escalation will follow a standard 3+3 escalation scheme. Evidence of HR-deficiency is not required to enroll in phase I, but is for phase II. All subjects will be treated with rucaparib (continuous oral administration twice daily) 400mg (dose level I), 500mg (II) or 600mg (III-IV) and intravenous copanlisib (D1, D8, D15; 28-day cycle; dose levels I-III: 45 mg; level IV: 60 mg). Once the RP2D of copanlisib and rucaparib in the phase I part of the study is determined, enrollment will commence into the phase II part of the trial. The primary objective of phase II is to estimate the preliminary efficacy of the combination of copanlisib and rucaparib among subjects with mCRPC carrying mutations in DNA repair genes, as measured by prostate-specific antigen (PSA) response. Up to 20 patients will enroll in the phase II using a Simon's two-stage design. In the first stage, 14 patients will be accrued. If there are 7 or fewer PSA responses in these 14 patients, the study will be stopped for futility. Otherwise, an additional 6 patients will be accrued. Patients with mCRPC need to have had disease progression during treatment with abiraterone, enzalutamide, and/or apalutamide. Prior therapy with taxanes is allowed. Translational objectives include the correlation of clinical outcomes with HR mutations and PI3K/mTOR pathway gene alterations in the tumor. Genomic analyses will also investigate biomarkers and mechanisms of action of the combination. The clinical trial is recruiting (NCT04253262). Citation Format: Andre De Souza, Rahul Aggarwal, Howard Safran, Dragan Golijanin, Roxanne Wood, Adam Olszewski, Wafik S. El-Deiry, Anthony Mega, Benedito A. Carneiro. BrUOG360: A phase Ib/II study of copanlisib combined with rucaparib in patients with metastatic castration-resistant prostate cancer (mCRPC) [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 CT222.

Targeting the PI3K and MAPK pathways to improve response to HER2-targeted therapies in HER2-positive gastric cancer

BackgroundAberrant PI3K signalling is implicated in trastuzumab resistance in HER2-positive gastric cancer (GC). The role of PI3K or MEK inhibitors in sensitising HER2-positive GCs to trastuzumab or in overcoming trastuzumab resistance is unclear.MethodsUsing mass spectrometry-based genotyping we analysed 105 hotspot, non-synonymous somatic mutations in PIK3CA and ERBB-family (EGFR, ERBB2, ERBB3 and ERBB4) genes in gastric tumour samples from 69 patients. A panel of gastric cell lines (N87, OE19, ESO26, SNU16, KATOIII) were profiled for anti-proliferative response to the PI3K inhibitor copanlisib and the MEK1/2 inhibitor refametinib alone and in combination with anti-HER2 therapies.ResultsPatients with HER2-positive GC had significantly poorer overall survival compared to HER2-negative patients (15.9 months vs. 35.7 months). Mutations in PIK3CA were only identified in HER2-negative tumours, while ERBB-family mutations were identified in HER2-positive and HER2-negative tumours. Copanlisib had anti-proliferative effects in 4/5 cell lines, with IC50s ranging from 23.4 (N87) to 93.8 nM (SNU16). All HER2-positive cell lines except SNU16 were sensitive to lapatinib (IC50s 0.04 µM–1.5 µM). OE19 cells were resistant to trastuzumab. The combination of lapatinib and copanlisib was synergistic in ESO-26 and OE-19 cells (ED50: 0.83 ± 0.19 and 0.88 ± 0.13, respectively) and additive in NCI-N87 cells (ED50:1.01 ± 0.55). The combination of copanlisib and trastuzumab significantly improved growth inhibition compared to either therapy alone in NCI-N87, ESO26 and OE19 cells (p < 0.05).ConclusionsPI3K or MEK inhibition alone or in combination with anti-HER2 therapy may represent an improved treatment strategy for some patients with HER2-positive GC, and warrants further investigation in a clinical trial setting.

Abstract OT-34-01: Phase Ib/II trial of copanlisib in combination with trastuzumab and pertuzumab after induction treatment of HER2 positive metastatic breast cancer with PIK3CA mutation or PTEN mutation

Abstract Background:The PI3K/Akt/mTOR pathway is a critical regulator of cell growth, survival, and metabolism in cancer. Its activation plays an important role in resistance to chemotherapy and HER2 targeted therapy. PIK3CA activating mutations and PTEN loss were reported in 30% and 16% of BOLERO-1 and 32% and 12% of BOLERO-3 patients, respectively. Exploratory analyses suggested that the addition of everolimus to trastuzumab and chemotherapy improved progression free survival (PFS) in patients with PIK3CA mutations and PTEN loss. In the phase III CLEOPATRA trial, while the combination of pertuzumab (P) plus trastuzumab (H) plus docetaxel (T) as compared with trastuzumab (H) plus docetaxel (T), significantly prolonged PFS (18.5 vs 12.4 months) for first-line treatment for HER2-positive (+ve) metastatic breast cancer (MBC), longer median PFS was observed in patients with wildtype versus mutated PIK3CA in both the control (13.8 v 8.6 months) and pertuzumab groups (21.8 v 12.5 months). Copanlisib is a highly selective, class 1 pan-PI3K inhibitor with predominant activity against both the δ and α isoforms. It is currently FDA approved for the treatment of adults with relapsed follicular lymphoma. This study hypothesizes that the addition of copanlisib to dual HER2 targeted therapy after first line induction treatment will improve clinical outcomes in HER2 positive MBC patients with PIK3CA or PTEN genomic alterations. Trial Design: This is a randomized, two- arm, open label, phase-2 study to evaluate the clinical activity of copanlisib added to HP maintenance after induction with THP in HER2 +ve MBC patients with PIK3CA mutations or PTEN loss. A safety run-in cohort (phase 1B) will be performed. Copanlisib will be administered weekly on D1, D8 of a 21-day cycle. Eligibility criteriaHER2 +ve MBC based on ASCO-CAP criteria (HER2 status based on metastatic tissue)• Activating mutations in PIK3CA, or PTEN loss• ECOG performance status ≤1• Normal organ and marrow function• Within 8 weeks of completion of first-line induction therapy with THP (Phase-2). Any prior treatment provided eligible to receive THP induction (Phase-1B) Specific aimsTo assess the benefit of adding copanlisib to HP in HER2+ve MBC patients with PIK3CA mutations or PTEN loss after induction treatment (Phase-2)• To determine safety and recommended phase 2 dose (RP2D) of copanlisib, HP combination in HER2 MBC patients (Phase-1B)• To correlate PFS and OS with the triplet combination with the number of induction cycles, hormone receptor status, and PTEN loss by IHC• To identify potential predictive and prognostic biomarkers for copanlisib activity Statistical methodsThe primary objective of the phase-1B portion is to determine the RP2D for the combination of copanlisib, trastuzumab, and pertuzumab. Phase 1 portion will use a 3+3 dose de-escalation design. The primary objective of the phase 2 portion is to determine a difference in PFS with the addition of copanlisib to HP maintenance after induction. Projected median PFS in control group is 8 months and 16 months in the experimental arm. We aim to detect a HR of 0.50 with power of 0.90 with 1-sided alpha of 0.1. With a sample size of 82, 12 months post-accrual follow-up, and accrual rate of 5 patients per month, the study duration is 30 months. To have 82 evaluable patients with a 15% drop-out rate, we would need to enroll 96 patients. A Wieand rule futility interim analysis will be conducted when half of the total of 54 required PFS events are observed. Citation Format: Senthil Damodaran, Rashmi K Murthy, Maliha Nusrat, Babita Saigal, Samantha C Trager, Debu Tripathy, Funda Meric-Bernstam. Phase Ib/II trial of copanlisib in combination with trastuzumab and pertuzumab after induction treatment of HER2 positive metastatic breast cancer with PIK3CA mutation or PTEN mutation [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-34-01.

Combination treatment of copanlisib and gemcitabine in relapsed/refractory PTCL (COSMOS): an open-label phase I/II trial

Current treatment options for peripheral T-cell lymphomas (PTCLs) in the relapsed/refractory setting are limited and demonstrate modest response rates with rare achievement of complete response (CR). This phase I/II study (NCT03052933) investigated the safety and efficacy of copanlisib, a phosphatidylinositol 3-kinase-α/-δ inhibitor, in combination with gemcitabine in 28 patients with relapsed/refractory PTCL. Patients received escalating doses of intravenous copanlisib on days 1, 8, and 15, administered concomitantly with fixed-dose gemcitabine (1000 mg/m2 on days 1 and 8) in 28-day cycles. Dose-limiting toxicity was not observed in the dose-escalation phase and 60 mg copanlisib was selected for phase II evaluation. Twenty-five patients were enrolled in phase II of the study. Frequent grade ≥3 adverse events (AEs) included transient hyperglycemia (57%), neutropenia (45%), thrombocytopenia, (37%), and transient hypertension (19%). However, AEs were manageable, and none were fatal. The overall response rate was 72% with a CR rate of 32%. Median duration of response was 8.2 months, progression-free survival was 6.9 months, and median overall survival was not reached. Combination treatment produced a greater CR rate in patients with angioimmunoblastic T-cell lymphoma than those with PTCL-not otherwise specified (55.6% versus 15.4%, respectively, P= 0.074) and progression-free survival was significantly longer (13.0 versus 5.1 months, respectively, P= 0.024). In an exploratory gene mutation analysis of 24 tumor samples, TSC2 mutation was present in 25% of patients and occurred exclusively in responders. The combination of copanlisib and gemcitabine is a safe and effective treatment option in relapsed/refractory PTCLs and represents an important new option for therapy in this rare group of patients.

Preliminary Results from a Phase II Study of Response-Adapted Therapy with Copanlisib and Rituximab for Untreated Follicular Lymphoma

Introduction Follicular lymphoma (FL) has a highly variable clinical course. Chemoimmunotherapy can induce durable remissions, but ~20% relapse early. The PI3K pathway is central to FL biology and multiple PI3K inhibitors (PI3Ki) are approved for FL, but the molecular profile of tumors most sensitive to PI3Ki is unknown. Further, PI3Ki are dosed indefinitely which contributes to toxicity and cost. Copanlisib is an IV inhibitor of PI3Kα and δ isoforms with high activity in relapsed FL. We hypothesized that patients with FL tumors most sensitive to PI3Ki will achieve deep and durable remissions after a fixed duration of copanlisib-based therapy. Here we report preliminary results of a response-adapted study using copanlisib and rituximab as frontline therapy for FL. Methods Pts with untreated grade 1-2, 3A FL, ≥stage 2, and any tumor burden are eligible if they meet criteria for need of systemic therapy that includes symptoms, increasing size of nodes, critical organ involvement, or impending organ compromise. No prior systemic therapy other than radiation is permitted. Frozen or archival tissue is required. Eligibility includes age ≥18 and adequate organ function unless involved by FL. Active HIV, CMV, Hep B or C, and autoimmune conditions requiring therapy are excluded. All pts receive PCP prophylaxis. Pts first receive copanlisib 60mg on days 1, 8, and 15 of a 28-day window to test activity of monotherapy. On-treatment tumor biopsies are optional after the window. Following the window, pts receive 6 cycles of copanlisib 60mg on days 1, 8, and 15 of a 28 day cycle along with rituximab 375mg weekly x 4 then on day 1 of each cycle. Streck tubes for circulating tumor DNA (ctDNA) are collected weekly during the window, after each cycle, and during surveillance. FDG-PET and CT scans are performed at baseline, after the window, and after cycles 3 and 6 to determine response. Patients with complete response (CR) after 6 cycles stop therapy. Patients with partial response (PR) after 6 cycles receive another 6 cycles of combination therapy with the copanlisib reduced to day 1 and 15 of each cycle. Non-responding patients will receive standard chemotherapy. The primary endpoint is the overall rate of CR with secondary endpoints of safety and duration of CR. Exploratory objectives include identification of a signature that predicts PI3Ki response. Results Ten pts have been enrolled and completed the copanlisib window. Median age was 50y (range, 28-77) including 3 (30%) over age 70. Seven (70%) pts had high-risk FLIPI scores ≥3 and two (20%) pts had Grade 3A FL. Comorbid conditions included prediabetes in 4 (40%) and hypertension in 4 (40%). All 10 (100%) pts had tumor reductions during copanlisib monotherapy with a median reduction of 41% (range 16-62%) (Figure 1). Four pts have completed 6 cycles of copanlisib and rituximab and all 4 (100%) have responded including 2 (50%) who achieved CR. In the two pts who achieved a PR after 6 cycles, one had a 90% tumor reduction and one had only persistent minimal residual disease in the bone marrow by flow cytometry. Toxicity was evaluated in 10 pts across 58 cycles and the most common have included rash (50%), diarrhea (50%) and mucositis (40%) which have all been G1 or G2 and successfully managed with supportive care and did not recur with subsequent cycles. One pt developed grade 3 neutropenia that responded to growth factors and did not recur. Four pts had dose delays due to rash (N=3) and lung infection (N=1). One pt had copanlisib reduced to 45mg due to recurrent rash and elevated liver tests. No pt has discontinued therapy. One pt required oral diabetic medications during therapy that were stopped after therapy completed. One pt had asymptomatic PCP pneumonia diagnosed during the copanlisib window prior to starting prophylaxis that was successfully treated while therapy continued. Conclusion Copanlisib is highly active in untreated FL and the first 10 (100%) patients all had tumor reductions after the first cycle of copanlisib monotherapy, including patients with high tumor bulk. Combination of copanlisib and rituximab can induce complete responses after only 6 cycles and without indefinite therapy. The safety profile includes rash and diarrhea that respond to supportive care and lessen on subsequent cycles. Updated clinical results from this ongoing trial (NCT03789240) along with the molecular profile of FL tumors will be presented at the meeting. Disclosures No relevant conflicts of interest to declare.

Combination of copanlisib with cetuximab improves tumor response in cetuximab-resistant patient-derived xenografts of head and neck cancer.

Despite recent advances, the treatment of head and neck squamous cell carcinoma (HNSCC) remains an area of high unmet medical need. HNSCC is frequently associated with either amplification or mutational changes in the PI3K pathway, making PI3K an attractive target particularly in cetuximab-resistant tumors. Here, we explored the antitumor activity of the selective, pan-class I PI3K inhibitor copanlisib with predominant activity towards PI3Kα and δ in monotherapy and in combination with cetuximab using a mouse clinical trial set-up with 33 patient-derived xenograft (PDX) models with known HPV and PI3K mutational status and available data on cetuximab sensitivity. Treatment with copanlisib alone resulted in moderate antitumor activity with 12/33 PDX models showing either tumor stabilization or regression. Combination treatment with copanlisib and cetuximab was superior to either of the monotherapies alone in the majority of the models (21/33), and the effect was particularly pronounced in cetuximab-resistant tumors (14/16). While no correlation was observed between PI3K mutation status and response to either cetuximab or copanlisib, increased PI3K signaling activity evaluated through gene expression profiling showed a positive correlation with response to copanlisib. Together, these data support further investigation of PI3K inhibition in HNSCC and suggests gene expression patterns associated with PI3K signaling as a potential biomarker for predicting treatment responses.

Abstract P1-19-24: A phase Ib trial of copanlisib in combination with trastuzumab in pretreated recurrent or metastatic HER2-positive breast cancer “PantHER”

Abstract Background Activation of the phosphoinositide -3 kinase (PI3K) pathway is a resistance mechanism to anti-HER2 targeted therapy. This trial was conducted to determine the maximum tolerated dose (MTD) of copanlisib, an oral pan-class I PI3K inhibitor, combined with trastuzumab for patients with advanced HER2 positive breast cancer resistant to anti-HER2 therapy. Patients and Methods In this phase Ib open label dose escalation study, using a 6 + 6 design, patients with advanced HER2-positive breast cancer who had disease progression following at least one prior line of HER2 therapy in the metastatic setting were treated with a dose escalation schedule of copanlisib (dose level 1 =45mg or dose level 2 = 60mg) IV on days 1, 8 and 15 of a 28 day cycle along with a fixed dose of trastuzumab 2mg/kg weekly after a loading dose of 4mg/kg in cycle 1. Archival tumour tissue, voluntary serial tumour biopsies and serial plasma samples were collected for genomic sequencing. Results Twelve patients were enrolled. MTD was determined as copanlisib 60mg plus trastuzumab 2mg/kg weekly. There was no dose limiting toxicity. The most common treatment-related adverse events (AE) of any grade experienced in more than 2 patients were hyperglycemia (58%), fatigue (58%), nausea (58%) and hypertension (50%). Confirmed stable disease at 16 weeks was observed in 6 (50%) participants. PIK3CA mutations were detected in archival tumour tissue of 6 (50%) patients and did not appear to influence likelihood of clinical benefit. PIK3CA mutations were detected in serial plasma ctDNA of all 12 patients and fluctuated over the course of treatment. Next-Generation Sequencing (NGS) analysis identified novel somatic mutations in the TTRAP gene, which encodes a PI3K-like protein kinase, detected only in tumour samples obtained at metastatic time points. Additionally, NGS analysis demonstrated clear temporal genomic heterogeneity with decreasing PIK3CA mutation variant allele frequency (VAF) post therapy Conclusions The combination of copanlisib and trastuzumab was safely administered with good overall tolerability in this trial. Preliminary anti-tumour stability was observed in patients with heavily pre-treated metastatic HER2 positive breast cancer. Translational studies identified a number of potential biomarkers for further study in the now initiated phase 2 clinical trial. Citation Format: Niamh M Keegan, Simon Furney, Janice Walshe, Giuseppe Gullo, John Kennedy, Kyran Bulger, John McCaffrey, Catherine M Kelly, Keith Egan, P O'Donovan, Andres Hernando, Ausra Teiserskiene, Imelda Parker, Angela M Farrelly, Aoife Carr, Giulio Calzaferri, Ray McDermott, Maccon M Keane, Liam Grogan, Oscar S Breathnach, Patrick G Morris, Sinead Toomey, Bryan T Hennessy. A phase Ib trial of copanlisib in combination with trastuzumab in pretreated recurrent or metastatic HER2-positive breast cancer “PantHER” [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-19-24.

Targeting PI3K and PLK1 to Overcome Ibrutinib-Venetoclax Resistance in Mantle Cell Lymphoma

BTK inhibitors such as ibrutinib and BCL-2 inhibitors such as venetoclax are two of the most effective therapies used to treat patients with refractory/relapsed mantle cell lymphoma (MCL). However, MCL patients treated with these therapies alone or in combination frequently develop therapeutic resistance, which results in poor clinical outcome. Therefore, targeting the signaling pathways responsible for therapeutic resistance and disease progression will potentially overcome primary and acquired resistance in ibrutinib-venetoclax-refractory/relapsed MCL. Our previous study on a MCL patient cohort comparing ibrutinib-sensitive and -resistant patients revealed that PI3K-ATK signaling is closely associated with ibrutinib resistance. Our recent single-cell transcriptomic profiling of another MCL patient cohort comparing ibrutinib responders and ibrutinib-venetoclax non-responders revealed that PI3K-AKT-mTOR signaling and the G2/M checkpoint were significantly upregulated in ibrutinib-venetoclax non-responders and were highly associated with disease progression. The constitutive PI3K-AKT-mTOR signaling in ibrutinib-resistant or ibrutinib-venetoclax dual-resistant MCL cells may be important in overcoming the G2/M checkpoint and promoting cell survival and cell proliferation. All PI3K isoforms - α, β, γ and δ - are expressed in all the MCL cell lines tested. Copanlisib is a pan-PI3K inhibitor, which targets all PI3K isoforms, and was FDA-approved to treat follicular lymphoma. Compared to idelalisib, a PI3Kδ inhibitor, copanlisib is more potent in targeting MCL cells in vitro with IC50 at a nanomolar range. Copanlisib at low dosage of 6 mg/kg/2d significantly inhibited in vivo tumor growth of an ibrutinib-venetoclax dual-resistant PDX model. PLK1, a central player in regulating G2/M transition, acts upstream of PI3K/AKT signaling via phosphorylating PTEN to cause a tumor-promoting metabolic state. Volasertib, a specific PLK1 inhibitor, is under a Phase III clinical trial for patients with acute myeloid leukemia. Volasertib dramatically arrested MCL tumor cells at the G2/M phase, which caused cell apoptosis at a low nanomolar range. Volasertib at low dosage of 10 mg/kg/week diminished in vivo tumor growth of the ibrutinib-venetoclax dual-resistant PDX model. More interestingly, the combination of copanlisib and volasertib induced synergistic effects in ibrutinib-resistant, venetoclax-resistant, and ibrutinib-venetoclax dual-resistant cell lines in vitro. Moreover, combined treatment with copanlisib (6mg/kg/2d) and volasertib (5mg/kg/week) significantly enhanced anti-MCL activity in the same PDX model, compared to that of either single-agent treatment. Our data demonstrated that pan-PI3K inhibitor copanlisib and PLK1 inhibitor volasertib are potent agents in targeting MCL cells in vitro and in vivo, and they have great potential to overcome ibrutinib and venetoclax resistance in MCL. When combined, copanlisib and volasertib have even greater potential to overcome ibrutinib and venetoclax resistance in MCL. Disclosures Wang: Dava Oncology: Honoraria; Juno Therapeutics: Research Funding; Aviara: Research Funding; Celgene: Honoraria, Research Funding; Loxo Oncology: Research Funding; VelosBio: Research Funding; BioInvent: Consultancy, Research Funding; Guidepoint Global: Consultancy; Kite Pharma: Consultancy, Research Funding; Acerta Pharma: Consultancy, Research Funding; MoreHealth: Consultancy, Equity Ownership; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau.

807P - Inhibition of the PI3K pathway in HER2-positive gastric cancer

BackgroundHER2 is overexpressed in 10-25% of gastric cancers (GCs). The association of PI3K pathway activating mutations with HER2 overexpression, or the impact of aberrant PI3K pathway signalling on response to trastuzumab (Tr) in GC has not been described. In HER2-positive breast cancer, targeting the PI3K pathway can overcome resistance to HER2-targeted therapies; however the role of PI3K inhibitors in sensitizing HER2-positive GCs to Tr or in overcoming Tr resistance is unknown. MethodsTumour samples from 69 GC patients were stratified into HER2-positive (n=29) and HER2-negative (n=40) groups. Mass spectrometry-based genotyping (Agena Bioscience) was used to analyse 105 hotspot somatic mutations in PIK3CA, EGFR, ERBB2, ERBB3 and ERBB4 in the tumours and in a panel of HER2-positive GC cell lines (N87, OE19, ESO26 and SNU16). The anti-proliferative response to the PI3K inhibitor copanlisib alone and in combination with the HER2-targeted therapies Tr and lapatinib was assessed in vitro. ResultsPatients with HER2-positive GC had significantly poorer overall survival compared to HER2-negative patients (15.9 months vs. 35.7 months; p=0.0032). PIK3CA, EGFR and ERBB mutations occurred more frequently in HER2-negative tumours than HER2-positive tumours, but had no impact on progression free or overall survival. In vitro OE19 cells were resistant to copanlisib, while all other cell lines were sensitive, independent of PIK3CA mutation status, with IC50s ranging from 23.4nm (N87) to 93.8nm (SNU16). All cell lines except SNU16 were sensitive to lapatinib with IC50s ranging from 0.04µM to 1.5µM. OE19 and SNU16 were resistant to Tr. The combination of lapatinib and copanlisib is synergistic in ESO-26 and OE-19 cells (ED50: 0.83±0.19 and 0.88±0.13, respectively) and additive in N87 cells (ED50:1.01±0.55). The combination of copanlisib and Tr significantly improved growth inhibition compared to either therapy alone in N87, ESO26 and OE19 cells (p<0.05). ConclusionsCopanlisib is an effective monotherapy in some HER2-positive GC cell lines. Combinations of copanlisib and Tr offer greater benefit than either drug alone, and may restore sensitivity to Tr in cells with intrinsic resistance to Tr. The addition of copanlisib to HER2 targeted therapy warrants further investigation in HER2-positive GC. Legal entity responsible for the studyThe authors. FundingNorth East Cancer Research and Education Trust (NECRET). DisclosureAll authors have declared no conflicts of interest.

Targeted Inhibition of PI3K α/δ Is Synergistic with BCL-2 Blockade in Genetically Defined Subtypes of DLBCL

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease that is transcriptionally classified into germinal center B-cell (GCB) and activated B-cell (ABC) subtypes. A subset of both GCB- and ABC-DLBCLs are dependent on B-cell receptor (BCR) signaling. Previously, we defined distinct BCR/PI3K-mediated survival pathways and subtype-specific apoptotic mechanisms in BCR-dependent DLBCLs (Cancer Cell 2013 23:826). In BCR-dependent DLBCLs with low baseline NF-κB activity (GCB tumors), targeted inhibition or genetic depletion of BCR/PI3K pathway components induced expression of the pro-apoptotic HRK protein. In BCR-dependent DLBCLs with high NF-κB activity (ABC tumors), BCR/PI3K inhibition decreased expression of the anti-apoptotic NF-κB target gene, BFL1.Our recent analyses revealed genetic bases for perturbed BCR/PI3K signaling and defined poor prognosis DLBCL subsets with discrete BCR/PI3K/TLR pathway alterations (Nat Med 2018 24:679). Cluster 3 DLBCLs (largely GCB tumors) exhibited frequent PTEN deletions/mutations and GNA13 mutations. Cluster 5 DLBCLs (largely ABC tumors) had frequent MYD88L265P and CD79B mutations that often occurred together. These DLBCL subtypes also had different genetic mechanisms for deregulated BCL2 expression - BCL2 translocations in Cluster 3 and focal (18q21.33) or arm level (18q) BCL2 copy number gains in Cluster 5. These observations prompted us to explore the activity of PI3K inhibitors and BCL2 blockade in genetically defined DLBCLs.We utilized a panel of 10 well characterized DLBCL cell line models, a subset of which exhibited hallmark genetic features of Cluster 3 and Cluster 5. We first evaluated the cytotoxic activity of isoform-specific, dual PI3Kα/δ and pan-PI3K inhibitors. In in vitro assays, the PI3Kα/δ inhibitor, copanlisib, exhibited the highest cytotoxicity in all BCR-dependent DLBCLs.We next assessed the transcriptional abundance of BCL2 family genes in the DLBCLs following copanlisib treatment. In BCR-dependent GCB-DLBCLs, there was highly significant induction of the pro-apoptotic HRK. In BCR-dependent ABC-DLBCLs, we observed significant down-regulation of the anti-apoptotic BFL1 protein and another NF-κB target gene, BCLxL (the anti-apoptotic partner of HRK).We then used BH3 profiling, to identify dependencies on certain BCL2 family members and to correlate these data with sensitivity to copanlisib. BCLxL dependency significantly correlated with sensitivity to copanlisib. Importantly, the BCLxL dependency was highest in DLBCL cell lines that exhibited either transcriptional up-regulation of HRK or down-regulation of BCLxL following copanlisib treatment.In all our DLBCL cell lines, PI3Kα/δ inhibition did not alter BCL2 expression. Given the genetic bases for BCL-2 deregulation in a subset of these DLBCLs, we next assessed the activity of the single-agent BCL2 inhibitor, venetoclax, in in vitro cytotoxicity assays. A subset of DLBCL cell lines was partially or completely resistant to venetoclax despite having genetic alterations of BCL2. We postulated that BCR-dependent DLBCLs with structural alterations of BCL2 might exhibit increased sensitivity to combined inhibition of PI3Kα/δ and BCL2 and assessed the cytotoxic activity of copanlisib (0-250 nM) and venetoclax (0-250 nM) in the DLBCL cell line panel. The copanlisib/venetoclax combination was highly synergistic (Chou-Talalay CI<1) in BCR-dependent DLBCL cell lines with genetic bases of BCL2 deregulation.We next assessed copanlisib and venetoclax activity in an in vivo xenograft model using a DLBCL cell line with PTENdel and BCL2 translocation (LY1). In this model, single-agent copanlisib did not delay tumor growth or improve survival. Single-agent venetoclax delayed tumor growth and improved median survival (27 vs 51 days, p<0.0001). Most notably, we found that the combination of copanlisib and venetoclax delayed tumor growth significantly longer than single-agent venetoclax (p<0.0001). Additionally, the combined therapy significantly increased survival in comparison with venetoclax alone (median survival 51 days vs not reached, p<0.0013).Taken together, these results provide in vitro and in vivo pre-clinical evidence for the rational combination of PI3Kα/δ and BCL2 blockade and set the stage for clinical evaluation of copanlisib/venetoclax therapy in patients with genetically defined relapsed/refractory DLBCL. DisclosuresLetai:AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Novartis: Consultancy, Other: Lab research report; Vivid Biosciences: Equity Ownership; AstraZeneca: Consultancy, Other: Lab research report. Shipp:AstraZeneca: Honoraria; Merck: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding.

Abstract 798: Pathway-directed high-throughput drug screen identifies PI3K inhibitors that synergistically potentiate antitumor activity of HDAC inhibitors in cutaneous T-cell lymphoma

Abstract Background: Cutaneous T-cell lymphoma (CTCL) represents a type of malignancy in the skin that is incurable. Recent studies demonstrated dysregulation of several signaling pathways in CTCL, including PI3K/AKT, JAK/STAT, and NFκB pathways. We performed a high-throughput drug screen to determine the potential of novel agents targeting these pathways for the treatment of CTCL. Methods: We compiled a library of 94 compounds targeting pathways known to be relevant in cancer biology, such as kinases involved in growth factor receptor signaling, HDACs, proteasome, DNA repair and regulators of apoptosis. The compounds were screened for antiproliferative activity against four CTCL cell lines in high-throughput proliferation assays. PI3K inhibitors were selected from the “hits” of the screen and were further studied in xenograft models of CTCL and in primary T-cell lymphomas cells from patients. To examine which PI3K isoform mediates antitumor effect in CTCL, isoform-specific siRNAs were used to knock down each isoforms as well as the combination of doublet isoforms. Based on this experiment, we tested antitumor activity of isoform-specific inhibitors. Promising candidates were also tested in combination assays using a matrix block method designed to detect synergistic activities. Results: From the high-throughput screen, we identified 14 compounds with antiproliferative activity in CTCL, including multiple inhibitors of the PI3K pathway. Validation assays confirmed the class effect of PI3K inhibitors in growth inhibition. From this class, a pan-PI3K inhibitor BKM120 was selected for in vivo studies. In a xenograft model of CTCL, BKM120 exhibited striking antitumor activity measured by a marked suppression of tumor growth and prolonged survival of tumor-bearing mice compared with vehicle control. To investigate which isoform of the PI3K mediates the antitumor effect in CTCL, we performed isoform-specific siRNA assays and demonstrated that knocking down isoform ð exhibited the most potent growth inhibition. Copanlisib targets α and δ isoforms of PI3K and is newly approved by the FDA for low-grade B-cell lymphoma. We demonstrated that copanlisib exhibits potent antiproliferation activity in CTCL cell lines. Furthermore, combination of copanlisib and LBH, a HDAC inhibitor, display synergistic antiproliferative effects across a broad dose gradient for each agent. Conclusion: A pan-PI3k inhibitor, BKM120, is highly active in CTCL cell lines and exnograft models. Inhibition of Isoform δ exhibits the most potent growth inhibition. Furthermore, an isoform-specific inhibitor, copanlisib, synergistically potentiates the effect of HDAC inhibitors against CTCL. These are highly promising approaches for the treatment of CTCL and warrant clinical investigation. Citation Format: Chi-Heng Wu, Chen-Yen Yang, Taha Rakhshandhroo, Laura Pincus, Sourav Bandyopadhyay, Frank McCormick, Weiyun Ai. Pathway-directed high-throughput drug screen identifies PI3K inhibitors that synergistically potentiate antitumor activity of HDAC inhibitors in cutaneous T-cell lymphoma [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 798.