pan-PI3K Inhibitor Research Articles

Biomimetic Small-Molecule Self-Assembly of PI3K inhibitor integrated with immunomodulator to amplify anticancer efficacy

Overactivation of PI3K signaling has been identified in prostate cancer. ZSTK is an effective pan-PI3K inhibitor, but it was withdrawn from early-phase clinical trials because of its poor solubility, on-target/off-tumor toxicity, and recruitment and polarization of tumor-associated macrophages (TAMs). Indomethacin (IND) reduces the polarization of TAMs, increases the proinflammatory macrophage (M1 phenotype) ratio in the tumor microenvironment, and enhances the immune response in tumor cells. Herein, we developed ZSTK carrier-free nanoparticle (ZNP)-loaded biomimetic hybrid indomethacin liposomes ([email protected]) with PC3 cell membranes (termed ZNPs/[email protected]). ZNPs/[email protected] displayed amplified synergistic therapeutic effects on zebrafish and nude mouse PC3 xenograft models. Their homologous targeting ability can deliver ZSTK to tumor tissue and elicit effective antiproliferative effects on PC3 cancer cells. Meanwhile, the released IND improved the immunosuppressive microenvironment, inducing immunological activation against cancer cells. Additionally, ZNPs/[email protected] did not cause liver toxicity compared with other treatment groups, as shown by histological analysis, suggesting their improved safety. Our data demonstrate that biomimetic hybrid liposome-coated carrier-free nanoparticles are a promising nanoplatform for amplifying the antitumor efficacy of kinase-inhibiting therapeutics with immunotherapy, but more preclinical and clinical evidence is required.

A phase I pharmacokinetic study of copanlisib in Chinese patients with relapsed indolent non-Hodgkin lymphoma

Copanlisib, a pan-PI3K inhibitor, has previously shown clinical efficacy and a tolerable safety profile in patients with indolent non-Hodgkin lymphoma. However, the pharmacokinetics, safety, tolerability, and efficacy of copanlisib in Chinese patients have not been reported. This was a single-arm, open-label, phase I study of copanlisib in Chinese patients with relapsed or refractory indolent non-Hodgkin lymphoma (iNHL). Patients received a single intravenous 60mg infusion of copanlisib over 1h on days 1, 8, and 15 of a 28-day cycle, with 1week of rest. Safety was monitored throughout the study, and plasma copanlisib levels were measured for pharmacokinetic analysis. Tumor response was determined by independent central radiologic review. Sixteen patients were enrolled and 13 were treated with 60mg of copanlisib for a median of 15.0weeks. With a Cmax of 566μg/L and a AUC (0-24) of 1880μg·h/L following single intravenous infusion, the pharmacokinetic parameters of copanlisib were consistent with that in previous studies, and no accumulation in plasma was observed. Treatment-emergent adverse events were reported for all 13 patients, the most common of which were hyperglycemia (100.0%), hypertension (76.9%), decreased neutrophil count (76.9%), and decreased white blood cell count (69.2%). Seven out of 12 evaluated patients achieved partial response, resulting in an overall response rate of 58.3% CONCLUSIONS: Copanlisib was well tolerated in Chinese patients with relapsed or refractory iNHL at the dose of 60mg and demonstrated encouraging disease control, thus warranting further clinical investigation. NCT03498430 (April 13, 2018).

ZDQ-0620, a Novel Phosphatidylinositol 3-Kinase Inhibitor, Inhibits Colorectal Carcinoma Cell Proliferation and Suppresses Angiogenesis by Attenuating PI3K/AKT/mTOR Pathway.

The PI3K/AKT pathway plays a central role in human cancers, aberrant activation of this pathway is associated with tumorigenesis, cancer progression and angiogenesis. Based on the importance of the PI3K/AKT pathway in malignancies, we developed a 4-aminoquinazoline derivative, ZDQ-0620, initially envisioned as a novel pan-PI3K inhibitor. This study aimed to evaluate the potential target of ZDQ-0620 and its anticancer effect in human colorectal carcinoma (CRC). PI3K-kinase activity test showed IC50 of ZDQ-0620 against PI3Ka was 0.5 nM; molecular docking, CETSA assay and western blotting was further performed to predict ZDQ-0620 was a PI3K/AKT pathway inhibitor by targeting PI3K. To identify the effect of ZDQ-0620 on CRC cells, Sulforhodamine B (SRB) assay, flow cytometry, and Cell morphology analysis were conducted. The results showed that ZDQ-0620 inhibited the proliferation, migration and invasion of CRC cells, induced apoptosis through G0/G1 cell cycle arrest and mitochondrial pathway. Additionally, ZDQ-0620 inhibited the migration and tube formation of human umbilical vein endothelial cells (HUVECs). In vivo, neovascularization of rat aortic ring and chick chorioallantoic membrane (CAM) induced by VEGF was diminished when treated with ZDQ-0620. These results indicate that ZDQ-0620 induce apoptosis and anti-angiogenesis via inhibits the PI3K/AKT pathway. We suggest that the great potential of ZDQ-0620 as an effective treatment candidate against CRC.

Abstract P4-01-07: Insights into the compensation mechanism behind targeting PI3K and Wnt in triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancer (TNBC) is a devastating disease accompanied with substantial morbidity and mortality with very few tools in the clinical armamentarium to improve outcomes. The PI3K pathway is activated in the majority of TNBCs. However, single-agent inhibition of PI3K has seen limited clinical efficacy. We have previously demonstrated that the Wnt pathway is upregulated in response to certain PI3K inhibitors as a compensatory resistance mechanism. Dual targeting of the PI3K and Wnt pathways leads to a synergistic anti-tumor effect that has been observed preclinically and in clinical trial (NCT03243331). Recently, we have found that several isoform-specific PI3K inhibitors predictably induce PTK7. PTK7 is a tyrosine kinase protein in the noncanonical Wnt pathway found to be overexpressed in various cancers, including TNBC, and is target of an antibody drug conjugate (ADC) currently in clinical trials. Herein, we sought to determine the PI3K isoforms that are responsible for PTK7 induction to help guide rational drug combination design. Methods: Twelve PI3K inhibitors including alpelisib, buparlisib, idelalisib, duvelisib, GSK2636771, GSK2292767, and CAY10505 were tested. Each inhibitor targets different isoforms individually or combinations of PI3K isoforms. Using four TNBC cell lines with different PI3K pathway mutational status and PTK7 expression (MDA-MB-231, MDA-MB-453, MDA-MB-468, and Hs578T), IC50s were initially measured using a viability assay. Using these IC50s, cells were treated in triplicate and RNA was isolated after 72 hours of treatment. RT-PCR was then performed using vehicle controls looking at the change in PTK7 expression. PI3K δ and λ isoform inhibition was also performed using siRNAs individually and in combination for validation of PI3K inhibitors. Protein expression of PI3K isoforms and PTK7 was also observed. Endogenous levels of PTK7 to assess induction after PI3K inhibitor treatment was assessed using both RNA and protein expression. Results: Testing multiple PI3K inhibitors covering various combinations of isoforms, we found that inhibition of PI3K δ and λ or pan-PI3K inhibition caused significant increases in RNA and protein levels of PTK7. We also found that individually targeting α and β in these cell lines did not have any effect on PTK7. We validated this induction further using siRNA inhibition to rule out any off targets of PI3K inhibitors. Additionally, after PI3K inhibition, the fold change in PTK7 expression was observed to be significantly higher in those cells with low or no PTK7 expression, whereas those with high baseline PTK7 expression observed little or no increase, maintaining pre-treatment levels. Conclusion: Refining clinical trial combinations and therapies to optimize drug efficacy while decreasing toxicity is a vital part of precision medicine. Here we have sought to optimize our previous clinical experience of dual targeting of PI3K and PTK7 and found two potential FDA approved therapies that may provide better efficacy and toxicities. Taking the previous trial and this preclinical data, future trials are warranted in TNBC. Citation Format: Jeffrey P Solzak, Chao Wang, Milan Radovich. Insights into the compensation mechanism behind targeting PI3K and Wnt in triple-negative breast cancer [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 P4-01-07.

The immune impact of PI3K-AKT pathway inhibition in colorectal cancer.

154 Background: Our prior work has shown that PI3K-altered colorectal cancer (CRC), with PIK3CA mutation or PTEN loss, has increased expression of key immune checkpoints (including PD-L1) resulting in immune evasion, despite increased immune engagement. Here, we investigated the impact of PI3K-AKT inhibition on the immune repertoire of CRC. Methods: Multiplex immunofluorescence was performed using two Vectra panels [1: AE1/AE3, CD3, CD8, PD-1, PD-L1, CD68; and 2: AE1/AE3, CD3, CD8, Granzyme B (GzB), CD45RO, FoxP3] on paired biopsies (baseline and cycle 1 day 15) from 6 patients with PI3K-altered metastatic CRC (mCRC) treated with AKT inhibitor, MK2206 (200 mg oral weekly), on a phase 2 clinical trial. Separately, one million CT26 CRC cells were implanted in BALB/C-e mice. After 48 hours, 10 mice/group were randomized for treatment with pan-PI3K inhibitor copanlisib (C, 10 mg/Kg IV 2x/week), anti-PD-1 (P, 200 µg IP 2x/week), copanlisib + anti-PD-1 (C+P), or control (Ct), for 21 days. Mouse tumors were stained with 6-plex immunohistochemistry (CD3, CD8, PD-L1, Ki67, GzB, AE1/AE3). Data were analyzed using related-samples Wilcoxon Signed-Rank test, Mann-Whitney U test, Kruskal-Wallis test, and Student’s t-test, as appropriate. Results: In PI3K-altered mCRC patients, AKT inhibition resulted in a trend towards increased median densities of intratumoral CD8+ T cells (0.8 vs 4.8 density/mm2, P = 0.14) and memory T cells (0 vs 10.3, P = 0.07), and decreased density of macrophages (12.4 vs 0, P = 0.07). No antigen experienced T cells were seen and activated CD8+ T cells were present in 1 patient only. In CT26 mice, PI3K and PD-1 co-inhibition resulted in the smallest mean tumor volumes (C+P 12% of Ct vs C 40% and P 42% of Ct, P < 0.05 for both), and the highest median % of intratumoral CD8+Ki67+ T cells as compared to all other treatment arms (C+P 1.6% vs C 0.5%, P 0.4%, Ct 0.6%, P < 0.05 for each pairwise comparison). C+P also increased the % of total CD3+ and CD8+ cells as compared to Ct and C (P < 0.05 for all). C alone did not increase immune infiltration in this non-PI3K activated model. Conclusions: PI3K-AKT pathway inhibition has the potential to improve effector T cell infiltration in PI3K-altered CRC. PI3K inhibitor synergizes with anti-PD-1 to improve treatment efficacy and CD8+ T cell proliferation. The mechanisms behind this immune repertoire shift are yet to be elucidated, such as via cytokine modulation. Therapeutic approaches to activate the proliferating CD8+ cells would be useful, and may require PI3Kα/β specific inhibitors to allow early T cell activation through PI3Kδ/γ isoforms.

Suppression of PI3K signaling is linked to autophagy activation and the spatiotemporal induction of the lens organelle free zone

The terminal steps of lens cell differentiation require elimination of all organelles to create a central Organelle Free Zone (OFZ) that is required for lens function of focusing images on the retina. Previous studies show that the spatiotemporal elimination of these organelles during development is autophagy-dependent. We now show that the inhibition of PI3K signaling in lens organ culture results in the premature induction of autophagy within 24 h, including a significant increase in LAMP1+ lysosomes, and the removal of lens organelles from the center of the lens. Specific inhibition of just the PI3K/Akt signaling axis was directly linked to the elimination of mitochondria and ER, while pan-PI3K inhibitors that block all PI3K downstream signaling removed all organelles, including nuclei. Therefore, blocking the PI3K/Akt pathway was alone insufficient to remove nuclei. RNAseq analysis revealed increased mRNA levels of the endogenous inhibitor of PI3K activation, PIK3IP1, in differentiating lens fiber cells preceding the induction of OFZ formation. Co-immunoprecipitation confirmed that PIK3IP1 associates with multiple PI3K p110 isoforms just prior to formation of the OFZ, providing a likely endogenous mechanism for blocking all PI3K signaling and activating the autophagy pathway required to form the OFZ during lens development.

Targeting the PI3K-AKT-mTOR Pathway in Castration Resistant Prostate Cancer: A Review Article.

Prostate cancer is one of leading causes of cancer death among men worldwide. Androgen deprivation therapy is a central part of the prostate cancer treatment algorithm, however, resistance to androgen deprivation commonly leads to disease progression. Mutations in the phosphoinositide-3-kinase pathway (PI3K) have been implicated in cancer progression and the development of castration-resistance. Thus, inhibitors of this pathway and its downstream signaling partners have been studied as potential therapeutic agents to treat metastatic castration resistant prostate cancer (mCRPC). In this article, we review recent clinical results for novel targeted therapies against the PI3K-AKT-mTOR pathway. Trials included in this systemic review were identified through conference abstracts, citations in review articles, PubMed, and ClinicalTrials.gov. Trial eligibility was independent of clinical setting or sample size. A total of 13 prospective clinical trials between 2012 and 2020 were reviewed: Two trials for pan-PI3K inhibitors, 2 trials for selective PI3K inhibitors, 4 trials for AKT inhibitors, 5 trials for mTOR inhibitors, and 1 for a combined PI3K and mTOR inhibitor. All studies were phase I or II studies with primary outcomes of either safety and tolerability or efficacy. Overall, pan-PI3K inhibitors and selective-PI3K inhibitors have not demonstrated clinical efficacy and may have significant adverse effects. AKT inhibitors may have significant adverse effects, but showed some evidence of improved survival. mTORC1 inhibitors show modest efficacy and significant adverse effects.

PI3K/AKT Activation Mediates B-Cell Transformation By the “T” Splice-Variant of Fyn Kinase

The SRC-family kinase FYN has key roles downstream of growth-factor receptor activation and is implicated in various cancers. Activating FYNutations were reported in angioimmunoblastic T-cell lymphoma (AITL), but its role in transforming lymphocytes is poorly characterized. FYN also may play a role in signaling downstream of the B cell Receptor (BCR) receptor in Diffuse Large B-Cell Lymphoma (DLBCL), and laboratory studies show inhibition of FYN and other SRC family kinases (SFKs) can block BCR-mediated lymphomagenesis. To investigate FYN's role in oncogenic singling, we introduced FYN alleles to IL3-dependent FL5.12 murine pro-B cells and tested for selective advantage upon cytokine withdrawal and ability to transform the cells to cytokine independence. FYN B and T variants result from alternate splicing of exon 7, with B originally detected in brain and T in hematopoietic cells, but both are expressed and may be deregulated associated with disease states in diffuse tissues. We employed wildtype (WT) alleles for both spliceoforms along with mutations detected recurrently in AITL (L174R and R176C), kinase active (Y528H), and kinase dead (K299A). No FYN B alleles showed any selective advantage in the prolymphocyte cells subjected to cytokine withdrawal. In contrast, all FYN T alleles other than kinase dead were strongly enriched through multiple rounds of IL3 withdrawal and rescue. Indeed, complete withdrawal of cytokine resulted in transformation to IL3-independent growth, generating stable cell lines addicted to FYN kinase activity for survival. The SFK inhibitor PP2 was toxic to all FYN-T transformed cells but not to baseline FL5.12 cells growing in cytokine. WT- and L147R-transformed cells were similarly sensitive to PP2, while the other AITL-derived mutant R176C demonstrated reduced sensitivity. Immunoblotting demonstrated PP2-sensitive phosphorylation of AKT and the MTORC1 target 4EBP1, suggesting PI3K/AKT activation as a key survival pathway downstream from transformation by FYN T alleles. Consistently, the AKT inhibitor ipatasertib and PDK1 inhibitor GSK2334470 were highly active against all FYN-transformed lines, and AKT phosphorylation was sensitive to PDK1 inhibition, showing convergence on AKT activation in all transformants. Pan-PI3K inhibition was active against FYN transformants, while the delta isoform-specific inhibitor idelalisib showed only partial activity, suggesting activation of multiple PI3K isoforms downstream of FYN activity. These results suggest that AKT, PI3K, and PDK1 are important targets downstream of FYN, and that the PI3K/AKT pathway is the key survival mechanism in lymphomagenesis mediated by FYN. We show that WT and activating mutants of the FYN T spliceoform specifically, but not FYN B, induce drug-sensitive B lymphocyte malignant transformation by activating PI3K/AKT/MTORC1 signaling. DisclosuresNo relevant conflicts of interest to declare.

Targeting Metabolic Vulnerabilities in Primary Effusion Lymphoma Using the Novel Nucleoside Analog 6-Eti

Although nucleoside analogues have been used effectively in the clinic for the treatment of a wide range of hematological malignancies, lack of response to currently available nucleoside analogues and drug resistance limit their utility. A rare but highly aggressive cancer is primary effusion lymphoma (PEL). Through high throughput screening, we have discovered a novel nucleoside analog, called 6-ethylthioinosine (6-ETI) as a potent and selective inhibitor of PEL, with little activity in other lymphomas tested. PEL is a rare B-cell non-Hodgkin's lymphoma characterized by lymphomatous effusions in body cavities. It is associated with Kaposi's sarcoma herpesvirus (KSHV/HHV-8) infection and occurs mainly in immunocompromised patients. PEL is known to frequently be resistant to conventional chemotherapy (CHOP and EPOCH) resulting in poor prognosis and a rather incurable disease. Our studies demonstrated that 6-ETI is a pro-drug activated by adenosine kinase (ADK), an enzyme that is overexpressed in PEL cell lines and primary PEL specimens, as well as other plasma cell malignancies, including plasmablastic lymphoma (PBL) and multiple myeloma (MM). The latter is also responsive to 6-ETI in vitro and in mouse models. 6-ETI induces S phase arrest and inhibits DNA synthesis. RNA sequencing of in vitro generated PEL resistant clones and CRISPR knock out of ADK (ADK KO), respectively, indicated that mutations or loss of expression of ADK renders cells resistant to treatment. This data demonstrates that ADK expression can be used as a predictive biomarker of response to 6-ETI, which can help identify which patients are more likely to respond to this treatment.We investigated which pathways are differentially regulated in sensitive and resistant cells to better delineate the mechanism of action of 6-ETI and to design effective combinatorial regimens and prevent resistance. We found that drug sensitivity was associated with AMPK activation and inhibition of PI3K/mTOR/p70S6K signaling. Little is known about the function of ADK in plasma cell neoplasms. Knock-out of this protein in PEL, or use of ADK chemical inhibitors, do not affect their viability. Thus, we used ADK KO cell lines to examine the role of ADK in these tumors and to determine if cells undergo adaptations that may contribute to 6-ETI resistance and represent potential vulnerabilities to combat it. We performed metabolic and transcriptomic profiling of wild type (WT) (6-ETI sensitive) and ADK KO (6-ETI resistant) cells to achieve a comprehensive assessment of all the metabolic perturbations and gene expression changes induced by knocking out ADK. We also treated these cells with 6-ETI to examine the effects in sensitive and resistant cells. This integrated analysis revealed that 6-ETI depletes sensitive PEL cells of their nucleotide pools accompanied by the downregulation of several genes in purine and pyrimidine biosynthesis pathways. We found that adenine supplementation rescues sensitive PEL cells from 6-ETI induced cytotoxicity, reverses p70S6K inhibition and restores DNA synthesis suggesting that purine metabolism is a critical mediator of 6-ETI induced cytotoxicity. Using seahorse bioenergetic assay, we show that ADK KO resistant cells have impaired mitochondrial respiration indicating that ADK plays a critical role in mitochondrial bioenergetics. Metabolic profiling of these ADK KO resistant cells showed that these cells have elevated levels of de novo pyrimidine metabolic intermediates. Metabolic flux through de novo pyrimidine is controlled by the rate limiting enzyme CAD. The activity of CAD is regulated by ribosomal protein S6 Kinase 1(S6K1) by phosphorylation at its (Ser1859) site. Using western blotting, we observed a striking increase of phosphorylation of CAD at its S6K1 site (Ser1859) in ADK KO cells compared to WT cells.This is the first to date study that characterizes the role of ADK in lymphomas. Our data indicates that ADK KO cells have undergone metabolic reprogramming to upregulate de novo pyrimidine biosynthesis and p70S6K signaling. Moreover, we found that 6-ETI synergizes with the pan PI3K inhibitor BKM120 highlighting nucleotide metabolism and PI3K/mTOR signaling as key therapeutic vulnerabilities targeted by this novel nucleoside analog. DisclosuresNo relevant conflicts of interest to declare.

Deciphering the Mechanisms of Osteoblast-Induced Resistance of Leukemic Stem Cell (LSC) in Ph+ CML: Role of PI3-Kinase, BRD4 and MYC and Development of Strategies to Overcome Osteoblast-Induced Resistance

Chronic myeloid leukemia (CML) is a stem cell neoplasms characterized by the chromosome translocation t(9;22) and the related BCR-ABL1 fusion gene. Therapy with BCR-ABL1 kinase inhibitors is highly effective in the treatment of CML and deep molecular responses are achieved in most patients. However, not all patients respond to these drugs due to resistance of leukemic stem cells (LSC). Recent data suggest that the disease-related microenvironment, known as the stem cell niche contributes to drug resistance and relapse in CML. So far, little is known about the resistance mechanisms protecting niche cells in the bone marrow of patients with CML. We have recently shown that osteoblasts are a major site of LSC-mediated resistance against BCR-ABL1-targeting drugs in CML. In the current study, we screened for drugs that are able to suppress growth and viability of osteoblasts and/or other niche-related cells and can thereby overcome drug resistance of CML LSC. Proliferation was analyzed by determining 3H-thymidine uptake in niche-related cells and apoptosis was measured by Annexin-V/DAPI-staining and flow cytometry. We found that the dual PI3 kinase (PI3K) and mTOR inhibitor BEZ235 and the selective pan-PI3K inhibitor copanlisib suppress proliferation of primary osteoblasts (BEZ235 IC 50: 0.05 µM; copanlisib IC 50: 0.05 µM), the osteoblastic cell line CAL-72 (BEZ235 IC 50: 0.5 µM; copanlisib IC 50: 1 µM), primary human umbilical vein endothelial cells (BEZ235 IC 50: 0.5 µM; copanlisib IC 50: 0.5 µM) and the endothelial cell line HMEC-1 (BEZ235 IC 50: 1 µM; copanlisib IC 50: 1 µM), whereas no comparable effects were seen with the mTOR inhibitor rapamycin. As determined by flow cytometry, BEZ235 and copanlisib suppressed the expression of phosphorylated (p) pAKT and pS6 in endothelial cells and osteoblasts whereas rapamycin downregulated pS6 expression but did not decrease expression of pAKT. Moreover, we found that BEZ235 and copanlisib cooperate with nilotinib and ponatinib in suppressing growth and viability of osteoblasts and endothelial cells. Furthermore, BEZ235 and copanlisib were found to overcome osteoblast-induced resistance of K562, KU812 cells, and primary CD34 +/CD38 − CML LSC against nilotinib and ponatinib. This effect was also seen when CAL-72 cells were first exposed to BEZ235 or copanlisib and washed before co-cultures with CML cells and BCR-ABL1 inhibitors were prepared, suggesting that osteoblast inhibition was a crucial event capable of disrupting LSC resistance in these co-cultures. Of all other drugs tested, only the BRD4-targeting drug JQ1 was found to suppress CAL72-induced resistance in the CML cell lines KU812 and K562, suggesting that osteoblast-induced resistance of CML cells is also mediated by a BRD4-MYC pathway. In a next step, we examined the expression of resistance-mediating immune checkpoint molecules on CML cells (KU812, K562, LSC) and on osteoblasts by flow cytometry. We found that CML cells and CAL72 cells constitutively express PD-L1 and that interferon-gamma (IFN-G) promotes the expression of PD-L1 in all cell types tested. Moreover, we found that the BRD4 blocker JQ1 and the BRD4-degrader dBET6 suppress the IFN-G-induced upregulation of PD-L1 in CML LSC and osteoblasts. In conclusion, our data show that osteoblast-induced resistance of CML stem cells is mediated by a PI3K-dependent pathway and BRD4/MYC, and that BRD4-inhibition or BRD4-degradation counteracts osteoblast-induced resistance of CML (stem) cells against BCR-ABL1 inhibitors and PD-L1 expression on CML LSC and osteoblasts. We hypothesize that checkpoint inhibition may assist in drug-induced eradication of CML LSC and thus in the development of curative drug therapies in Ph + CML. DisclosuresHoermann: Novartis: Honoraria. Gleixner: Pfizer: Honoraria; Abbvie: Honoraria; BMS: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Sperr: AbbVie, BMS-Celgene, Daiichi Sankyo, Deciphera, Incyte, Jazz, Novartis, Pfizer, StemLine, Thermo Fisher: Honoraria, Research Funding. Valent: Novartis: Honoraria; Pfizer: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; OAP Orphan Pharmaceuticals: Honoraria.

Synergistic antitumor activity of pan-PI3K inhibition and immune checkpoint blockade in bladder cancer

BackgroundImmune checkpoint blockade (ICB) induces durable response in approximately 20% of patients with advanced bladder urothelial cancer (aUC). Over 50% of aUCs harbor genomic alterations along the phosphoinositide 3-kinase (PI3K)...

Development of PI3K inhibitors: Advances in clinical trials and new strategies (Review)

Phosphatidylinositol 3-kinases (PI3Ks) are the family of vital lipid kinases widely distributed in mammalian cells. The overexpression of PI3Ks leads to hyperactivation of the PI3K/AKT/mTOR pathway, which is considered a pivotal pathway in the occurrence and development of tumors. Hence, PI3Ks are viewed as promising therapeutic targets for anti-cancer therapy. To date, some PI3K inhibitors have achieved desired therapeutic effect via inhibiting the activity of PI3Ks or reducing the level of PI3Ks in clinical trials, among which, Idelalisib, Alpelisib and Duvelisib have been approved by the FDA for treatment of ER+/HER2- advanced metastatic breast cancer and refractory chronic lymphocytic leukemia (CLL) and small lymphocytic lymphomas (SLL). This review focuses on the latest advances of PI3K inhibitors with efficacious anticancer activity, which are classified into Pan-PI3K inhibitors, isoform-specific PI3K inhibitors and dual PI3K/mTOR inhibitors based on the isoform affinity. Their corresponding structure characteristics and structures-activity relationship (SAR), together with the progress in the clinical application are mainly discussed. Additionally, the new PI3K inhibitory strategy, such as PI3K degradation agent, for the design of potential PI3K candidates to overcome drug resistance is referred as well.

530P Phase II study of PI3K inhibitor copanlisib in cancer patients with deleterious PTEN mutations and retained PTEN protein expression: Results from the NCI-MATCH Trial (EAY131) sub-protocol Z1H

The NCI-MATCH trial, the largest national study (1173 sites) for patients with relapsed/refractory solid tumors, lymphomas and myeloma, assigns targeted therapies based on individual tumor molecular alterations detected using clinical targeted next-generation sequencing and immunohistochemistry. It was hypothesized that patients with deleterious PTEN mutations without PTEN protein loss may benefit from treatment with the pan-PI3K inhibitor copanlisib.

Defining the therapeutic selective dependencies for distinct subtypes of PI3K pathway-altered prostate cancers

Previous studies have suggested that PTEN loss is associated with p110β signaling dependency, leading to the clinical development of p110β-selective inhibitors. Here we use a panel pre-clinical models to reveal that PI3K isoform dependency is not governed by loss of PTEN and is impacted by feedback inhibition and concurrent PIK3CA/PIK3CB alterations. Furthermore, while pan-PI3K inhibition in PTEN-deficient tumors is efficacious, upregulation of Insulin Like Growth Factor 1 Receptor (IGF1R) promotes resistance. Importantly, we show that this resistance can be overcome through targeting AKT and we find that AKT inhibitors are superior to pan-PI3K inhibition in the context of PTEN loss. However, in the presence of wild-type PTEN and PIK3CA-activating mutations, p110α-dependent signaling is dominant and selectively inhibiting p110α is therapeutically superior to AKT inhibition. These discoveries reveal a more nuanced understanding of PI3K isoform dependency and unveil novel strategies to selectively target PI3K signaling nodes in a context-specific manner.

Apoptin Overexpression Efficiently Amplified Cytotoxic Effects of PI3K Inhibition Using BKM120 in Lymphoblastic Leukemia Cell Lines.

Purpose: Although the complex structure of acute lymphoblastic leukemia (ALL) and involvement of diverse pathways in its pathogenesis have put an obstacle in the way of efficient treatments, identification of strategies to manipulate the genome of neoplastic cells has made the treatment prospective more optimistic. Methods: To evaluate whether the transduction of apoptin __a gene encoding a protein that participates in the induction of apoptosis__ could reduce the survival of leukemic cells, we generated recombinant lentivirus expressing apoptin, and then, MTT assay, flow cytometric analysis of DNA content, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were applied. Results: Transduction of apoptin into different leukemic cells was coupled with the reduction in the viability and proliferative capacity of the cells. Among all tested cell lines, Nalm-6 and C8166 were more sensitive to the anti-leukemic property of apoptin. Moreover, we found that the transduction of apoptin in the indicated cell lines not only induced G2/M cell cycle arrest but also induced apoptotic cell death by altering the balance between pro- and anti-apoptotic target genes. The efficacy of apoptin transduction was not limited to these findings, as we reported for the first time that the overexpression of this gene could potentiate the anti-leukemic property of pan PI3K inhibitor BKM120. Conclusion: The results of this study showed that the transduction of apoptin into lymphoblastic leukemia cell lines induced cytotoxic effects and enhanced therapeutic value of PI3K inhibition; however, further investigations are demanded to ascertain the safety and the efficacy of apoptin transduction in patients with ALL.

Abstract 367: Sub-group of ovarian cancer patients with hyperactive RAS network signaling identified: Dynamic pathway activity test identifies patients that may benefit from PI3K/mTOR or PI3K/mTOR/BCL inhibitors

Abstract Background: Dysregulated RAS signaling through GPCRs is reported in ovarian cancer (OC), but no sub-groups of OC patients with actionable RAS genetic variants have been found. The complex mechanisms that link RAS nodes can trigger adaptation when a single RAS node is inhibited. Thus, inhibition of multiple RAS nodes may be required for durable antitumor responses. To identify patients with dysregulated RAS signaling tumors that may respond to RAS node inhibitors, an assay using an impedance biosensor was developed. The CELsignia RAS Activity Test measures GPCR-initiated signaling activity and PI3K, mTOR, and BCL's role in transducing this activity in live tumor cells. The current study set out to characterize the prevalence of dysregulated RAS signaling initiated by a GPCR in OC patients and the role played by PI3K, mTOR and BCL. Methods: Fresh tumor specimens from 32 OC patients and 18 OC cell lines (84% HGS, 13% endometrioid, 3% clear cells) were used to derive test cell samples. Dynamic live cell response to a GPCR agonist (LPA), a PI3K-α inhibitor (GDC-0077), a pan-PI3K inhibitor (copanlisib), a pan-PI3K/mTOR inhibitor (gedatolisib), and a BCL inhibitor (navitoclax) was measured using an xCELLigence impedance biosensor (Agilent Technologies). From these responses, the gross amount of GPCR-initiated signaling and corresponding participation of PI3K-α, all Class 1 PI3K-isoforms, mTORC1, and BCL was quantified and converted to a signaling score. Correlative analyses using FACS markers (RPS6, AKT, ERK, caspase 3) were performed. Results: Hyperactive RAS signaling (RASs+) initiated by GPCR pathways was found in 14 of 50 (28%; 95% CI=17%-42%) tumors. The signaling scores were bimodally distributed; for the 14 RASs+ and the 36 RASs- patient tumors, the mean scores were 569 (SD 282) and 55 (SD 78), respectively. For RASs+ tumors, inhibition of all Class 1 PI3K-isoforms attenuated, on average, 57% of the GPCR signaling, while inhibition of PI3K-α alone had only a nominal inhibitory effect. When mTOR inhibition was combined with pan-PI3K inhibition, GPCR signaling was further attenuated by 29%. More complete attenuation of RAS-signaling occurred when PI3K, mTOR, and BCL were simultaneously inhibited. Early apoptosis markers (RPS6, AKT, ERK, caspase 3) were found most significantly in RASs+ tumors with pan-PI3K/mTOR inhibition and to a greater degree with pan-PI3K/mTOR/BCL inhibition. No apoptosis markers were found in RASs- tumors regardless of which RAS node was inhibited. Conclusions: These findings suggest that a significant sub-group of OC patients have a RAS-involved oncogenic driver that is responsive ex vivo to pan-PI3K/mTOR and pan-PI3K/mTOR/BCL inhibitors. A clinical trial to evaluate treatment response of this patient sub-group to combined PI3K/mTOR or PI3K/mTOR/BCL inhibitors is warranted. Citation Format: Stefano Rossetti, Aaron Broege, Joanna Sabat, Shu Wiley, Salmaan Khan, Catherine Kuzmicki, Ian MacNeil, Brian Sullivan, Lance Laing. Sub-group of ovarian cancer patients with hyperactive RAS network signaling identified: Dynamic pathway activity test identifies patients that may benefit from PI3K/mTOR or PI3K/mTOR/BCL inhibitors [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 367.

Abstract 1377: Volume scanning, a rational approach to covalent PI3Kα inhibitors

Abstract Inhibitors of the phosphatidylinositol 3-kinase (PI3K) - protein kinase B (PKB/Akt) - mechanistic target of rapamycin (mTOR) axis are considered valuable assets in cancer therapy. A considerable effort has been dedicated to the development of drugs targeting class I PI3Ks, which are evaluated in preclinical and clinical studies.[1-5] Here we present a strategy to convert a phase II clinical candidate, a pan-PI3K inhibitor (PQR309, bimiralisib)[5,6], into a highly selective, covalent PI3Kα inhibitor with the aim to minimize off-target and on-target metabolic side effects of PI3K inhibitor cancer therapy. We exploited a rational approach to increase target selectivity by covalently targeting PI3Kα at the non-conserved nucleophilic Cys862. A combination of warhead activity design, proximity screening and an optimized orientation allowed a tight control of reversible inhibitor binding in combination with an isoform-specific covalent reaction. To avoid off-target reactions, all warheads' reactivities were determined and optimize for selectivity and of Cys862 modification. An extensive Structure Activity Relationship (SAR) study was performed and a wide range of linear and restricted rotation linkers were introduced. A comprehensive understanding of the kinetics of irreversible inhibition acquired by kinetic TR-FRET assays and subsequent determination of kchem, kinact and calculated Ki allowed the establishment of a SAR, for compound selection with minimal off-target reactivity and high PI3Kα selectivity. X-ray crystallography and MS-based proteomics validated the covalent modification of Cys862. Our pilot compounds exceed specificity and potency over an experimental dimethyl-substituted enone, CNX-1351.[7] Moreover, our compounds display increased stability in rat liver microsomal assays and outperform the rapidly metabolized CNX-1351. Our strategy to investigate and tune warheads' reactivity represents a major step forward in the rational design of covalent chemical tools, overcoming the serendipity in the discovery of irreversible compounds. Moreover, we provide highly selective chemical tools to dissect PI3K isoform signaling in physiology and disease. A clarification of the role of the different PI3K isoforms in insulin signaling allows to address the challenges in isoform selectivity and to develop PI3K inhibitors showing ideal isoform specificity. [1] Rageot D et. al. J Med Chem. 2019, 62 (13), 6241-6261. [2] Borsari C et. al. ACS Med Chem Lett. 2019, 10 (10), 1473-1479. [3] Marone R et al. Biochim Biophys Acta 2008, 1784 (1), 159-185. [4] Wymann MP and Schneiter R Nat Rev Mol Cell Biol. 2008, 9 (2), 162-176. [5] Beaufils F et. al. J Med Chem. 2017, 60 (17), 7524-7538. [6] Wicki A et. al. Eur J Cancer. 2018, 96, 6-16. [7] Nacht M et. al. J Med Chem. 2013, 56 (3), 712-721. Citation Format: Chiara Borsari, Erhan Keles, Jacob McPhail, Alexander Schäfer, Rohitha Sriramaratnam, Martina De Pascale, Matthias Gstaiger, John Burke, Matthias P. Wymann. Volume scanning, a rational approach to covalent PI3Kα inhibitors [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 1377.

Abstract 342: Sub-group of HER2-negative breast cancer patients with hyperactive RAS network signaling identified: Dynamic pathway activity test identifies patients that may benefit from PI3K/mTOR or PI3K/mTOR/BCL inhibitors

Abstract Background: Dysregulated signaling through GPCRs is implicated in oncogenic RAS signaling in breast cancer (BC). The complex regulatory mechanisms that link RAS nodes can trigger an adaptive response (e.g. resistance) when a single RAS node is inhibited. Inhibition of multiple RAS nodes may thus be required to achieve durable antitumor responses. To identify patients with dysregulated RAS signaling tumors that may respond to RAS node inhibitors, an assay using an impedance biosensor was developed. The CELsignia RAS Activity Test measures GPCR-initiated signaling activity and PI3K, mTOR, and BCL's role in transducing this activity in live tumor cells. The current study set out to characterize the prevalence of dysregulated RAS signaling initiated through a GPCR in HER2-negative BC patients and the role played by PI3K, mTOR and BCL. Methods: Fresh tumor specimens from 69 HER2- BC patients were used to derive tumor cell samples. Dynamic live cell response to a GPCR agonist (LPA), a PI3K-α inhibitor (GDC-0077), a pan-PI3K inhibitor (copanlisib), a pan-PI3K/mTOR inhibitor (gedatolisib), and a BCL inhibitor (navitoclax) was measured using an xCELLigence impedance biosensor (Agilent Technologies). From these responses, the gross amount of GPCR-initiated signaling and corresponding participation of PI3K-α, all Class 1 PI3K-isoforms, mTORC1, and BCL was quantified and converted to a signaling score. Correlative analyses using FACS markers (RPS6, AKT, ERK, caspase 3) were performed. Results: Hyperactive RAS signaling (RASs+) initiated by GPCR pathways was found in 16 of 69 (23.1%; 95% CI=15%-34%) tumors. The signaling scores were bimodally distributed; for the 16 RASs+ and the 53 RASs- patient tumors, the mean scores were 486 (SD 248) and 91 (SD 73), respectively. For the 16 RASs+ tumors, inhibition of all Class 1 PI3K-isoforms attenuated, on average, 71% of the GPCR signaling, while inhibition of PI3K-α alone had only a nominal inhibitory effect. When mTOR inhibition was combined with pan-PI3K inhibition, GPCR signaling was further attenuated by 14%. The most complete attenuation of RAS-signaling occurred when PI3K, mTOR, and BCL were simultaneously inhibited. Early apoptosis markers (RPS6, AKT, ERK, caspase 3) were found most significantly in RASs+ tumors with pan-PI3K/mTOR inhibition and to a greater degree with pan-PI3K/mTOR/BCL inhibition. No apoptosis markers were found in RASs- tumors regardless of which RAS node was inhibited. Conclusions: These findings suggest that a significant sub-group of BC patients have a RAS-involved oncogenic driver that is responsive ex vivo to pan-PI3K/mTOR and pan-PI3K/mTOR/BCL inhibitors. A clinical trial to evaluate treatment response of this patient sub-group to combined PI3K/mTOR or PI3K/mTOR/BCL inhibitors is warranted. Citation Format: Salmaan Khan, Adrish Sen, Catherine Kuzmicki, Ian MacNeil, Aaron Broege, Sarah Mutka, Kelly Brass, Ky McCracken, Laura Milligan, Katja Kotke, Brian Sullivan, Lance Laing. Sub-group of HER2-negative breast cancer patients with hyperactive RAS network signaling identified: Dynamic pathway activity test identifies patients that may benefit from PI3K/mTOR or PI3K/mTOR/BCL inhibitors [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 342.

Abstract 1378: A novel, highly potent PI3Kα covalent inhibitor deconvolutes class I PI3K isoforms in cancer cells

Abstract Phosphoinositide 3-kinase (PI3K) imposes central regulatory signals that promote cell growth, proliferation, motility, and suppression of apoptosis. PI3K is dysregulated in a broad spectrum of human tumours.[1] Considering the central role attributed to PI3K signaling in cancer biology, the success of clinical trials in the field was below expectations, mainly due to metabolic on-target adverse effects. Here, we present a novel, highly selective PI3Kα-covalent inhibitor (compound 19), which combines the exceptional affinity of the pan-PI3K inhibitor PQR514[2] with a linker moiety, directing a warhead to selectively react via a Michael addition with isoform-specific Cys862 of PI3Kα. Cellular assay platforms were combined to validate 19, specifically, to assess its target selectivity and occupancy, and to translate its in vitro activity to cellular readouts. Overall, 19 exhibited excellent cellular activity with a superior profile compared to CNX-1351[3], a previously reported covalent PI3Kα inhibitor. A NanoBRET (Bioluminescence Resonance Energy Transfer) drug-target engagement assay allowed for an investigation of inhibitory efficacy in HEK293 cells. NanoBRET experiments exploiting the Cys862Ser mutant PI3Kα protein confirmed selective reactivity with Cys862. Various cell lines either constitutively activated in PI3Kα (MCF7, T47D, SKOV3) or deficient in PTEN (PC3 and A2058) were used to resolve target engagement, which confirmed a persistent and prolonged suppression of protein kinase B (PKB/Akt, Ser473) phosphorylation after washout of 19 over non-covalent structural analogs thereof that lack a functional warhead. Furthermore, nucleo-cytoplasmic translocation probes based on the FOXO1 transcription factor enabled the investigation of signal propagation downstream of PKB/Akt. In PI3Kα-dependent cell lines, FOXO1, maintained a predominant nuclear localization even after removal of 19, confirming extended PI3Kα targeting upon covalent attachment of specifically this drug. Importantly, 19 provided a considerable gain in potency over BYL719, a clinically approved non-covalent PI3Kα-selective inhibitor. Taken together, our results present a major step towards an increased local and temporal control of PI3Kα signaling through isoform-selective covalent anchoring. This strategy is a step towards the development of drug-like, isoform-selective, covalent PI3K inhibitor scaffolds, and 19 provides a template for the development of PI3Kα inhibitors with overall reduced adverse side effects in cancer therapy. Moreover, our covalent inhibition strategy will pave the way for a better molecular understanding of PI3K isoform signaling in general. [1] Wymann M. P. and Schneiter R. Nat. Rev. Mol. Cell Biol. 2008, 9, 162-176. [2] Borsari, C et. al. ACS Med. Chem. Lett. 2019, 10 (10), 1473-1479. [3] Nacht M et. al. J. Med. Chem. 2013, 56 (3), 712-721. Citation Format: Erhan Keles, Chiara Borsari, Rohitha Sriramaratnam, Thorsten Schäfer, Matthias P. Wymann. A novel, highly potent PI3Kα covalent inhibitor deconvolutes class I PI3K isoforms in cancer cells [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 1378.

Resistance Mechanisms Underlying Venetoclax Resistance in Mantle Cell Lymphoma

Background: BCL-2 is highly expressed in mantle cell lymphoma (MCL), and targeting BCL-2 with venetoclax (ABT-199) has resulted in positive clinical outcomes in MCL patients. However, resistance to various targeted agents often emerges; therefore, we generated venetoclax-resistant MCL cell lines to uncover the mechanisms mediating acquired venetoclax resistance and performed high-throughput drug screening to identify novel anti-MCL agents that can overcome venetoclax resistance.Methods: We generated 3 venetoclax-resistant cell lines (Mino, Rec-1 and Granta519) by long-term venetoclax exposure, and the anti-MCL activity of venetoclax against these acquired resistance lines was measured with dose-dependent cell viability and apoptosis assays compared with the parental lines. Aberrant protein expression between the parental and resistant cell lines was detected using Reverse Phase Protein Array (RPPA) analysis. A drug library (> 1000 drugs; Selleck Chemicals Ltd) was screened to identify agents that inhibit the cell growth of both the parental and resistant cells. The candidate drugs that showed anti-MCL activity were confirmed by dose-dependent cell viability and Annexin V apoptosis assays. To determine the mechanisms underlying the anti-MCL activity of the candidate agents in the parent and venetoclax-resistant MCL cell lines, we conducted RPPA analysis before and after drug treatment.Results: Chronic exposure to venetoclax resulted in markedly less sensitivity to venetoclax. The IC50 values of the venetoclax-resistant cell lines (Mino-Re, Rec-1-Re and Granta519-Re) were determined to be 603 nM, 676 nM and 456 nM, respectively, which are 600-fold, 500-fold and 25-fold higher than the IC50 values of the parental cells lines. RPPA analysis showed that proteins such as PTEN, PDCD4, AKT, ATM, BCL-2, BAX and BAK were down-regulated and p-AKT, p21, STAT3, Cyclin B1 and MCL-1 were up-regulated in the venetoclax-resistant MCL cell lines. The results were confirmed by Western blotting, and further Western blotting experiments revealed expression and modification differences among anti-apoptotic and pro-apoptotic protein family members between the parental and venetoclax-resistant MCL cell lines. Specifically, both MCL-1 and BCL-xL were elevated; however, BIM and BAX were absent in the venetoclax-resistant cells. High-throughput drug screening showed striking anti-MCL activity with two PI3K inhibitors, PIK-75 (PI3K α inhibitor) and GSK1059615 (pan PI3K inhibitor), in dose-dependent cell viability and apoptosis assays. RPPA analysis of the parental and venetoclax-resistant MCL cell lines with and without PI3K inhibitor treatment suggested that both drugs inhibited the cell viability of the MCL cell lines by blocking the activated PI3K/AKT pathway.Conclusion:The PI3K/AKT pathway is activated in venetoclax-resistant MCL cell lines, and targeting this pathway may be a potential approach to overcoming venetoclax resistance. DisclosuresWang:Pharmacyclics: Research Funding; Onyx: Research Funding; Kite Pharma: Research Funding; Dava Oncology: Honoraria; Celgene: Honoraria, Research Funding; BeiGene: Research Funding; Asana Biosciences: Research Funding; Proteolix: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Research Funding; Acerta Pharma: Consultancy, Research Funding.