Dual PI3K Inhibitor Research Articles

Cancer Therapy with Quinoxaline Derivatives: Dual Inhibition of Pi3k and Mtor Signaling Pathways

Quinoxaline is a heterocyclic compound with a benzene ring fused to a pyrazine ring. It learned much about his ability to cure many diseases, including cancer. Phosphoinositide 3kinase (PI3K) and the mTOR (mammalian target of rapamycin) are the 3 essential pathways which regulate the growth, survival, and development of cells. Dysregulation of such pathways is often seen in cancer, making them attractive targets for cancer therapy. In particular, several quinoxaline derivatives show promise as two inhibitors of the mTOR/PI3K signalling pathway, which is often dysregulated in cancer along with many other diseases. One such example is PX866, which has shown activity against several cancers in previous studies. In previous studies, another quinoxaline derivative, PKI587, also showed strong inhibitory activity against mTOR and PI3K. PKI587 was researched in several clinical trials for treating many cancer types, which include non-small cell lung as well as breast cancer. Dual inhibitors of mTOR and PI3K, including quinoxaline derivatives, inhibit cell growth and cancer by blocking the signalling of these two important factors. In recent years, quinoxaline derivatives have emerged as potent dual inhibitors of PI3K and mTOR, and in this review, we explore the latest developments in this area.

Dual PI3K/mTOR inhibitor PF-04979064 regulates tumor growth in gastric cancer and enhances drug sensitivity of gastric cancer cells to 5-FU

Gastric cancer (GC) is characterized by high tumor heterogeneity, increased surgical difficulty, and limited chemotherapy efficacy, and it is associated with a poor prognosis. The abnormal proliferation of cells involves abnormal activation of the PI3K/AKT/mTOR signaling pathway. Inhibition of this signaling pathway can inhibit tumor cell proliferation and induce cell apoptosis. This study evaluated the effect of PF-04979064, a dual inhibitor of PI3K and mTOR, on human GC cells. PF-04979064 significantly inhibited the proliferation of human gastric adenocarcinoma AGS cells and the undifferentiated GC cell line HGC-27, promoting cell apoptosis. Combination treatment with PF-04979064 and the GC first-line clinical drug 5-FU showed synergistic effects, and PF-04979064 markedly increased the sensitivity of GC cells to chemotherapy drugs. Western blot results showed that PF-04979064 significantly inhibited the PI3K/AKT/mTOR signaling pathway in GC cells, whereas RNA seq results demonstrated substantial alterations in gene expression profiles upon treatment with PF-04979064. This study provides insight into the effects of PF-04979064, thereby establishing a solid foundation for its potential clinical application in the treatment of GC.

Inhibition of PI3K/Akt/mTOR Signaling Pathway Suppresses 5-Fluorouracil Resistance in Gastric Cancer.

At present, 5-Fluorouracil (5-FU) is a crucial anti-cancer drug and is widely used for the treatment of various carcinomas, including gastric cancer (GC). The resistance of GC cells to 5-FU is still a matter of great concern. To illustrate the role of PI3K/Akt/mTOR signaling in regulating the cell cycle progression and migration of 5-FU-resistant GC cells. After the establishment of drug-resistant GC cell lines, the effects of 5-FU and/or BEZ235 (the dual inhibitor of PI3K and mTOR) on the activity of parental or drug-resistant GC cells were explored. The viability and localization of GC cells (MKN-45 and MKN-74) and their drug-resistant cells (MKN-45/R and MKN-74/R) were assessed using MTT assays and immunofluorescence staining. The impacts of 5-FU and/or BEZ235 on GC cell cycle progression and cell migration were assessed via flow cytometry analyses and wound healing assays, respectively. GC tissues were collected from patients with GC sensitive or refractory to 5-FU chemotherapy. RT-qPCR and western blot were conducted to measure PI3K, AKT, and mTOR levels in GC cells or tissues. After 5-FU treatment, GC cells displayed 5-FU resistance and the viability of drug-resistant cells (MKN-45/R and MKN-74/R) was higher than that of parental cells (MKN-45 and MKN-74). The IC50 values for MKN-45 and MKN-45/R were 8.93 ug/ml and 140 ug/ml, and the values for MKN-74 and MKN-74/R were 3.93 ug/ml and 114.29 ug/ml. Additionally, the PI3K/Akt/mTOR signaling pathway was activated in drug-resistant GC cells and tumor tissues of patients refractory to 5-FU chemotherapy, as evidenced by high PI3K, Akt, and mTOR levels in MKN-45/R, MKN-74/R, and GC tissues resistant to 5-FU. BEZ235 promoted cell cycle arrest and suppressed the migration of GC cells. Moreover, the combination of BEZ235 and 5-FU led to more effective suppressive influence on cell cycle progression and cell migration relative to the single 5-FU or BEZ235 treatment. Silencing of the PI3K/Akt/mTOR signaling pathway suppressed the 5-FU resistance of GC cells.

Inhibition of PI3K/Akt/mTOR signaling by NDRG2 contributes to neuronal apoptosis and autophagy in ischemic stroke

BackgroundAstrocytic N-myc downstream-regulated gene 2 (NDRG2), a differentiation- and stress-associated molecule, has been involved in the cause of ischemic stroke (IS). However, its downstream effector in IS remains unclear. This study aimed to characterize expression of NDRG2 in IS patients and rats and to investigate the underlying mechanism. MethodsThe protein expression of NDRG2 and mammalian target of the rapamycin (mTOR) and the extent of mTOR phosphorylation in plasma of IS patients were detected by ELISA. An oxygen-glucose deprivation model was established in mouse neuronal cells CATH.a, followed by cell counting kit-8, flow cytometry, TUNEL, and western blot assays to examine cell viability, apoptosis and autophagy. Finally, the effect of NDRG2-mediated phosphatidylinositol 3-kinase/protein kinase-B/mTOR (PI3K/AKT/mTOR) pathway on neuronal apoptosis and autophagy was verified in rats treated with middle cerebral artery occlusion. ResultsNDRG2 was highly expressed in the plasma of IS patients, while the extent of mTOR phosphorylation was reduced in IS patients. NDRG2 blocked the PI3K/Akt/mTOR signaling through dephosphorylation. Depletion of NDRG2 suppressed apoptosis and autophagy in CATH.a cells, which was reversed by a dual inhibitor of PI3K and mTOR, BEZ235. In vivo experiments confirmed that NDRG2 promoted neuronal apoptosis and autophagy by dephosphorylating and blocking the PI3K/Akt/mTOR signaling. ConclusionThe present study has shown that NDRG2 impairs the PI3K/Akt/mTOR pathway via dephosphorylation to promote neuronal apoptosis and autophagy in IS. These findings provide potential targets for future clinical therapies for IS.

Identification of phytochemical as a dual inhibitor of PI3K and mTOR: a structure-based computational approach.

Breast cancer is a common form of cancer that affects both men and women. One of the most common types of genomic flaws in cancer is the aberrations in the PI3K/AKT/mTOR pathway. The benefit of dual targeting PI3K as well as mTOR is that the kinase-positive feedback loops are more effectively inhibited. Therefore, in the current study, structure-based models like molecular docking, MM-GBSA, Qikprop, induced fit docking, simulated molecular dynamics (MD), and thermal MM-GBSA were used to identify the phytochemicals from the zinc 15 database, which may inhibit PI3K and mTOR. After docking the phytochemicals with PI3K (PDB 4FA6), ten ligands based on the docking score were selected, among which salvianolic acid C had the highest docking score. Hence, salvianolic acid A was also docked. All the ligands taken showed a binding energy of greater than -30kcal/mol. The predicted ADME showed that the ligands have druggable properties. By performing MD of the top five ligands and salvianolic acid A, it was found that ZINC000059728582, ZINC000257545754, ZINC000253532301, and salvianolic acid A form a stable complex with PI3K protein, among which ZINC000014690026 showed interaction with Val 882 for more than 89% of the time. Salvianolic acid A is already proven to suppress tumor growth in acute myeloid leukemia by inhibiting PI3K/AKT pathway, but the exact protein target is unknown. Therefore, the present study identifies new molecules and provides evidence for salvianolic acid A for dual inhibition. Further experiments must be performed both in vitro and in vivo to support the predictions of these computational tools.

FGF1 protects FGFR1-overexpressing cancer cells against drugs targeting tubulin polymerization by activating AKT via two independent mechanisms.

Cancer drug resistance is a common, unpredictable phenomenon that develops in many types of tumors, resulting in the poor efficacy of current anticancer therapies. One of the most common, and yet the most complex causes of drug resistance is a mechanism related to dysregulation of tumor cell signaling. Abnormal signal transduction in a cancer cell is often stimulated by growth factors and their receptors, including fibroblast growth factors (FGFs) and FGF receptors (FGFRs). Here, we investigated the effect of FGF1 and FGFR1 activity on the action of drugs that disrupt tubulin polymerization (taltobulin, paclitaxel, vincristine) in FGFR1-positive cell lines, U2OS stably transfected with FGFR1 (U2OSR1) and DMS114 cells. We observed that U2OSR1 cells exhibited reduced sensitivity to the tubulin-targeting drugs, compared to U2OS cells expressing a negligible level of FGFRs. This effect was dependent on receptor activation, as inhibition of FGFR1 by a specific small-molecule inhibitor (PD173074) increased the cells’ sensitivity to these drugs. Expression of functional FGFR1 in U2OS cells resulted in increased AKT phosphorylation, with no change in total AKT level. U2OSR1 cells also exhibited an elevated MDR1 and blocking MDR1 activity with cyclosporin A increased the toxicity of paclitaxel and vincristine, but not taltobulin. Analysis of tubulin polymerization pattern using fluorescence microscopy revealed that FGF1 in U2OSR1 cells partially reverses the drug-altered phenotype in paclitaxel- and vincristine-treated cells, but not in taltobulin-treated cells. Furthermore, we showed that FGF1, through activation of FGFR1, reduces caspase 3/7 activity and PARP cleavage, preventing apoptosis induced by tubulin-targeting drugs. Next, using specific kinase inhibitors, we investigated which signaling pathways are responsible for the FGF1-mediated reduction of taltobulin cytotoxicity. We found that AKT kinase is a key factor in FGF1-induced cell protection against taltobulin in U2OSR1 and DMS114 cells. Interestingly, only direct inhibition of AKT or dual-inhibition of PI3K and mTOR abolished this effect for cells treated with taltobulin. This suggests that both canonical (PI3K-dependent) and alternative (PI3K-independent) AKT-activating pathways may regulate FGF1/FGFR1-driven cancer cell survival. Our findings may contribute to the development of more effective therapies and may facilitate the prevention of drug resistance in FGFR1-positive cancer cells.

Co-targeting of HDAC, PI3K, and Bcl-2 results in metabolic and transcriptional reprogramming and decreased mitochondrial function in acute myeloid leukemia

Despite the recently approved new therapies, the clinical outcomes of acute myeloid leukemia (AML) patients remain disappointing, highlighting the need for novel therapies. Our lab has previously demonstrated the promising outlook for CUDC-907, a dual inhibitor of PI3K and HDAC, in combination with venetoclax (VEN), against AML both in vitro and in vivo at least partially through suppression of c-Myc. In this study, we further elucidated the mechanism of action of the combination in preclinical models of AML. We demonstrated that the combination significantly reduced primary AML cell engraftment in immunocompromised mice. RNA sequencing and metabolomics analyses revealed that the combination reduced the levels for mRNAs of key TCA cycle genes and metabolites in the TCA cycle, respectively. This was accompanied by a reduced oxygen consumption rate (OCR), demonstrating that the combination suppressed oxidative phosphorylation (OXPHOS). Metabolomics analyses revealed that a large number of metabolites upregulated in AraC-resistant AML cells could be downregulated by the combination. CUDC-907 synergized with VEN in inducing apoptosis in the AraC-resistant AML cells. In conclusion, the CUDC-907 and VEN combination induces metabolic and transcriptomic reprograming and suppression of OXPHOS in AML, which provides additional mechanisms underlying the synergy between the two agents.

Treatment by PI3K/mTOR Inhibitor BEZ235 Combined with TLR-7/8 Agonist Interfere with Immune Evasion Mechanisms of WEHI-3 Mouse Leukemia Cells.

Several PI3K/Akt/mTOR pathway inhibitors and TLR agonists induce tumor cell death. However, the mechanisms of these therapeutic approaches in acute myeloid leukemia (AML) cells are still unknown. To investigate the effects of BEZ235, as a dual inhibitor of PI3K and mTOR pathways, and TLR7/8 agonist R848 on the expression and regulation of the immune inhibitory molecules in myeloid leukemia cells. WEHI-3 leukemia cells were incubated with dual PI3K and mTOR inhibitor BEZ235 and TLR7/8 agonist R848 for 48 hrs. Firstly, cell viability was assessed by MTT method. The semi-quantitative relative mRNA expression of Galectin-9 (Gal-9), PD-L1, PVR, and STAT3 was assessed according to HPRT as a housekeeping gene. Finally, the protein expression of phosphorylated STAT3 was evaluated by western blotting analysis. WEHI-3 cells showed growth inhibition following treatment with BEZ235 and R848 whose combination exerted more proliferation arrest. The mRNA expression of Gal-9, PD-L1 and PVR immune checkpoint molecules significantly reduced in treated cells with BEZ235 and R848. Combined treatment indicated more reduction compared with the single treatment. Finally, the expression and phosphorylation of STAT3 were down-regulated after a single or dual treatment with BEZ235 and R848. Our results conclude that treatment with the combination of BEZ235 and R848 interferes with immune evasion mechanisms through STAT3-signaling pathway in WEHI-3 leukemia cells.

PI3K inhibitor 3-MA promotes the antiproliferative activity of esomeprazole in gastric cancer cells by downregulating EGFR via the PI3K/FOXO3a pathway

Gastric cancer is a common gastrointestinal malignancy worldwide, with a high mortality rate and poor prognosis. Esomeprazole (ESO) has been shown to have anticancer activity by affecting cell growth and autophagy and its mechanism in gastric cancer cells is evident. The PI3K/AKT/FOXO3a pathway is central in cancers. 3-Methyladenine (3-MA), a dual inhibitor of PI3K and autophagy, plays a synergistic role in combination with antitumor agents. In this study, we assessed the role of ESO on the PI3K/AKT/FOXO3a pathway and the beneficial effects of ESO combined with 3-MA in gastric cancer cells. Cell viability, proliferation, invasion, migration, apoptosis, autophagy, and protein expression were detected by CCK-8, EdU, Transwell, flow cytometry, immunofluorescence assay, and western blot. ESO decreased cell viability in a concentration- and time-dependent manner and increased autophagy with upregulation of LC3II and P62. Additionally, ESO inhibited the proliferation, migration, and invasion and induced the apoptosis of gastric cancer cells in a concentration-dependent manner. ESO inhibited PI3K/AKT/FOXO3a signaling and EGFR and SKP2 expression concentration-dependent. 3-MA enhanced the antiproliferative activity of ESO and synergistically inhibited PI3K/FOXO3a signaling and the expression of EGFR but not SKP2. Furthermore, pretreatment with the EGFR inhibitor AG1478 enhanced the antiproliferative activity of ESO in gastric cancer cells. In conclusion, our results suggested that the PI3K inhibitor 3-MA promotes the antiproliferative activity of ESO in gastric cancer cells by synergistically downregulating EGFR via the PI3K/FOXO3a pathway.

Identifying pathways regulating the oncogenic p53 family member \u0394Np63 provides therapeutic avenues for squamous cell carcinoma

BackgroundΔNp63 overexpression is a common event in squamous cell carcinoma (SCC) that contributes to tumorigenesis, making ΔNp63 a potential target for therapy.MethodsWe created inducible TP63-shRNA cells to study the effects of p63-depletion in SCC cell lines and non-malignant HaCaT keratinocytes. DNA damaging agents, growth factors, signaling pathway inhibitors, histone deacetylase inhibitors, and metabolism-modifying drugs were also investigated for their ability to influence ΔNp63 protein and mRNA levels.ResultsHaCaT keratinocytes, FaDu and SCC-25 cells express high levels of ΔNp63. HaCaT and FaDu inducible TP63-shRNA cells showed reduced proliferation after p63 depletion, with greater effects on FaDu than HaCaT cells, compatible with oncogene addiction in SCC. Genotoxic insults and histone deacetylase inhibitors variably reduced ΔNp63 levels in keratinocytes and SCC cells. Growth factors that regulate proliferation/survival of squamous cells (IGF-1, EGF, amphiregulin, KGF, and HGF) and PI3K, mTOR, MAPK/ERK or EGFR inhibitors showed lesser and inconsistent effects, with dual inhibition of PI3K and mTOR or EGFR inhibition selectively reducing ΔNp63 levels in HaCaT cells. In contrast, the antihyperlipidemic drug lovastatin selectively increased ΔNp63 in HaCaT cells.ConclusionsThese data confirm that ΔNp63-positive SCC cells require p63 for continued growth and provide proof of concept that p63 reduction is a therapeutic option for these tumors. Investigations of ΔNp63 regulation identified agent-specific and cell-specific pathways. In particular, dual inhibition of the PI3K and mTOR pathways reduced ΔNp63 more effectively than single pathway inhibition, and broad-spectrum histone deacetylase inhibitors showed a time-dependent biphasic response, with high level downregulation at the transcriptional level within 24 h. In addition to furthering our understanding of ΔNp63 regulation in squamous cells, these data identify novel drug combinations that may be useful for p63-based therapy of SCC.

Dual Targeting of PI3K and HDAC by CUDC-907 Inhibits Pediatric Neuroblastoma Growth.

The dysregulation of PI3K, HDACs, and MYCN are well known for promoting multiple cancer types, including neuroblastoma (NB). Targeting the upstream regulators of MYCN, including HDACs and PI3K, was shown to suppress cancer growth. In the present study, we analyze different NB patient datasets to reveal that high PI3K and HDAC expression is correlated with overall poor NB patient survival. High PI3K level is also found to be associated with high MYCN level and NB stage progression. We repurpose a dual inhibitor CUDC-907 as a single agent to directly target both PI3K and HDAC in NB. We use in vitro methodologies to determine the efficacy and selectivity of CUDC-907 using six NB and three control fibroblast cell lines. Our results show that CUDC-907 significantly inhibits NB proliferation and colony growth, induces apoptosis, blocks cell cycle progression, inhibits MYCN, and enhances H3K9Ac levels by inhibiting the PI3K/AKT signaling pathway and HDAC function. Furthermore, CUDC-907 significantly inhibits NB tumor growth in a 3D spheroid tumor model that recapitulates the in vivo tumor growth. Overall, our findings highlight that the dual inhibition of PI3K and HDAC by CUDC-907 is an effective therapeutic strategy for NB and other MYC-dependent cancers.

Abstract 2463: Enhancing the antileukemic activity of venetoclax against leukemia stem cells by targeting oxidative phosphorylation through dual inhibition of PI3K and HDAC

Abstract Over 40% of acute myeloid leukemia (AML) patients experience relapse after remission from intensive chemotherapy and stem cell transplant. Therefore, novel therapies are in demand to target the root cause of relapse, leukemia stem cells (LSCs). Recently, a selective inhibitor of anti-apoptotic Bcl-2, venetoclax, was FDA approved for treating AML in combination with less intensive therapies for patients with comorbidities. However, anti-apoptotic Mcl-1 overexpression and pro-apoptotic Bim downregulation manifests venetoclax resistance. Our previous work demonstrated that a novel dual inhibitor of PI3K and HDAC, CUDC-907, synergizes with venetoclax by overcoming these resistance mechanisms in bulk AML cells and suppressing c-Myc expression and mTOR activity. Previous literature has reported that Bcl-2 inhibition suppresses oxidative phosphorylation (OXPHOS), which is what LSCs rely on for energy production. This is in contrast to normal HSCs that can readily shift their reliance to glycolysis for ATP production if OXPHOS is inhibited. Since c-Myc and mTOR signaling are also known to support mitochondrial respiration, we hypothesize that the combination of CUDC-907 and venetoclax can selectively target LSCs. Metabolomics analyses revealed that pre-treatment of CUDC-907 followed by venetoclax affects the abundance of intermediates in the TCA cycle of AML cells, prior to apoptosis. RNAseq analyses with the same treatment regimen found that the transcription of the TCA cycle regulator pyruvate dehydrogenase kinase 2 (PDK2) is upregulated, and the transcription of pyruvate carboxylase (PC), aconitase 2 (ACO2) and the glutamine transporter SLC1A5 is downregulated, some of which are known cMyc target genes. These findings were correlated with a reduced oxygen consumption rate, indicating the suppression of OXPHOS. Treatment of patient-derived xenograft (PDX) mice with venetoclax and CUDC-907 reduced primary AML cell engraftment, suggesting both bulk AML cell and LSC suppression. These results suggest that the combination of CUDC-907 with venetoclax may target LSCs by interfering with the TCA cycle. To further establish our hypothesis, C13 glucose flux analysis will assist in following the effected metabolic pathways. Rescue experiments supplementing metabolites that were downregulated in treated AML cells and/or inhibition of PDK2 will confirm the mitochondrial respiration based mechanism of AML cell death by the combination of CUDC-907 and venetoclax. Finally we will confirm that these two drugs when combined, reduce the number of LSCs in primary patient samples with a PDX model and flow cytometry for known LSC markers, CD38, CD34 & CD123. The results of this study will form a solid foundation for the clinical evaluation of this promising combination therapy for the treatment of AML and potentially reduce the incidence of relapse. Citation Format: Katie Hege, Hasini Kalpage, Maik Hüttemann, Holly Pitman, Jeffrey W. Taub, Lisa Polin, Jing Li, Yubin Ge. Enhancing the antileukemic activity of venetoclax against leukemia stem cells by targeting oxidative phosphorylation through dual inhibition of PI3K and HDAC [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 2463.

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.

The combination of CUDC-907 and gilteritinib shows promising in vitro and in vivo antileukemic activity against FLT3-ITD AML

About 25% of patients with acute myeloid leukemia (AML) harbor FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations and their prognosis remains poor. Gilteritinib is a FLT3 inhibitor approved by the US FDA for use in adult FLT3-mutated relapsed or refractory AML patients. Monotherapy, while efficacious, shows short-lived responses, highlighting the need for combination therapies. Here we show that gilteritinib and CUDC-907, a dual inhibitor of PI3K and histone deacetylases, synergistically induce apoptosis in FLT3-ITD AML cell lines and primary patient samples and have striking in vivo efficacy. Upregulation of FLT3 and activation of ERK are mechanisms of resistance to gilteritinib, while activation of JAK2/STAT5 is a mechanism of resistance to CUDC-907. Gilteritinib and CUDC-907 reciprocally overcome these mechanisms of resistance. In addition, the combined treatment results in cooperative downregulation of cellular metabolites and persisting antileukemic effects. CUDC-907 plus gilteritinib shows synergistic antileukemic activity against FLT3-ITD AML in vitro and in vivo, demonstrating strong translational therapeutic potential.

A novel 4-aminoquinazoline derivative, DHW-208, suppresses the growth of human breast cancer cells by targeting the PI3K/AKT/mTOR pathway

Breast cancer is one of the most frequent cancers among women worldwide. However, there is still no effective therapeutic strategy for advanced breast cancer that has metastasized. Aberrant activation of the PI3K/AKT/mTOR pathway is an essential step for the growth of human breast cancers. In our previous study, we designed and synthesized DHW-208 (2,4-difluoro-N-(5-(4-((1-(2-hydroxyethyl)-1H-pyrazol-4-yl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)benzenesulfonamide) as a novel pan-PI3K inhibitor. This study aimed to assess the therapeutic efficacy of DHW-208 in breast cancer and investigate its underlying mechanism. We found that DHW-208 inhibited the growth, proliferation, migration, and invasion of breast cancer cells. Moreover, DHW-208 induced breast cancer cell apoptosis via the mitochondrial pathway and induced G0/G1 cell-cycle arrest. In vitro results show that DHW-208 is a dual inhibitor of PI3K and mTOR, and suppress the growth of human breast cancer cells by targeting the PI3K/AKT/mTOR pathway. Consistent with the in vitro results, in vivo studies demonstrated that DHW-208 elicits an antitumor effect by inhibiting the PI3K/AKT/mTOR-signaling pathway with a high degree of safety in breast cancer. Above all, we report for the first time that DHW-208 suppressed the growth of human breast cancer cells by inhibiting the PI3K/AKT/mTOR-signaling pathway both in vivo and in vitro. Our study may provide evidence for the use of DHW-208 as an effective, novel therapeutic candidate for the treatment of human breast cancers in clinical trials.

Phase Ib trial combining rapid determination of drug-drug interaction (DDI) followed by a dose finding period to assess safety and preliminary efficacy of fimepinostat plus venetoclax in patients with aggressive B-cell lymphoma.

8056 Background: Fimepinostat (F), a dual inhibitor of PI3K (α, β, δ) and HDACs type 1/2, causes suppression of MYC levels (Shulman et al., 2017). A pooled analysis of diffuse large B-cell lymphoma (DLBCL) pts treated with F in two Ph 1/2 trials revealed an objective response rate in evaluable/ITT MYC-altered DLBCL pts of 29%/23%, respectively (Landsburg et al., 2018). Based on compelling preclinical activity of F in combination with venetoclax (V) (a Bcl2 inhibitor), we initiated a Ph 1b/2 study of F + V in pts with relapsed or refractory (R/R) DLBCL or high-grade B-cell lymphoma (HGBL), with or without MYC alteration. V is metabolized primarily by CYP3A, and preclinical studies showed that F may inhibit CYP3A4 (IC50 of 13.58 and 0.28 µM for midazolam and testosterone, respectively), suggesting F could cause DDI with V. Methods: Cohorts of patients received increasing dose levels of F administered on a 5-days-on/2-days-off (5/2) schedule in combination with daily V in 21-day cycles (Cohort 1: F 30 mg QD 5/2 + V 400 mg QD; Cohort 2: F 60 mg QD, 5/2 + V 400 mg QD; Cohort 3: F 60 mg QD 5/2 + V 800 mg QD). A potential DDI was assessed during Cycle 0 (Table), where PK for V (10 mg) monotherapy was compared to that for V (10 mg) in the presence of F. Patient PK samples were collected, analyzed and reviewed in < 10 days to determine the final ramp-up dose level of V. Results: As of 1-Feb-2020, 16 pts have been enrolled in 2 dose cohorts. Intensive PK analysis of 13 pts showed only mild (≤ 2-fold) to no increase in V exposure in the presence of F. In Cohort 1 (n = 6), the mean AUC increased 1.6-fold, and mean Cmax by 1.5-fold. In Cohort 2 (n = 7), no increase in mean AUC (0.9-fold) or Cmax (1.0-fold) was observed. Accordingly, all pts ramped up V to 100% of the target dose (400 mg) upon entering Cycle 1; rapid escalation of V was well tolerated. DLT was observed in 1 pt (Grade 3 diarrhea) in Cohort 2. Overall, 75% of TEAEs have been mild or moderate (Grade 1/2), and most were of limited duration. 11 pts (69%) experienced SAEs; 4 pts (25%) had SAEs considered related to either F or V. Conclusions: Real-time PK evaluation showed that F had only a mild to no DDI with V. F + V is well tolerated at clinically active dose levels, and evaluation of higher dose-level cohorts was ongoing. Enrollment in Cohort 2 remains on-going. Clinical trial information: NCT01742988 . [Table: see text]

Correlation of MYC gene signature variations with clinical response to fimepinostat in patients with aggressive B-cell lymphoma.

e15654 Background: Fimepinostat (F), a small molecule dual inhibitor of PI3K α, β, and δ isoforms and Class I and II histone deacetylases (HDACs), is currently under investigation for the treatment of aggressive hematological cancers. Preclinical studies demonstrated that F both decreases transcription of myc and MYC-targeted genes (via HDAC inhibition) and increases destruction of MYC protein (via PI3K inhibition). This study sought to identify differences in transcript-level signatures of gene networks in pre-treatment tumor samples associated with future clinical response to F. Methods: Among 105 patients (pts) with relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) treated in phase 1/2 clinical trials of F (± rituximab), 33 pts had pre-treatment tumor biopsy samples available for molecular testing. All pts had measurable disease at baseline and had at least 1 post-baseline tumor assessment. To be included in the analysis set, pts must have been on study for ≥42 days. RNA libraries were created and sequenced on the Illumina HiSeq platform. Gene Set Enrichment Analysis (GSEA; Subramanian et al., 2005) was used to study changes in transcript expression patterns that correlate with responder (complete/partial response) or non-responder (progressive disease) pt populations. ~20,000 genes were rank-ordered by log2-fold change and tested against the MSigDB (Liberzon et al., 2011) collection of gene expression signatures. Results: > 1500 Gene Sets with False Discovery Rate (FDR) < = 0.05 and Normalized Enrichment Score (NES) > 1.3 for genes upregulated in the F-responder population were identified. These Gene Sets represent transcriptional activity associated with well characterized biological pathways or gene expression signatures determined empirically. The top 20 signatures by NES were associated with general transcription functions (E2F, RNA polymerase), chromosomal structure (meiotic recombination, telomere maintenance) and specifically, MYC transcription targets (NES: 2.7; FDR q-val: 0). Additional signatures consistent with PI3K (GSK3b) and HDAC activity in the responder population were also identified, as would be expected for a dual inhibitor of PI3Ks and HDACs. Conclusions: GSEA analysis of genes upregulated in the F-treated responder DLBCL pts are consistent with preclinical data showing that F functions to suppress MYC in tumor cells. F is currently undergoing active clinical study in combination with venetoclax for the treatment of R/R DLBCL or high grade B-cell lymphoma (HGBL): NCT01742988.

Phase Ib Trial of the PI3K Inhibitor Copanlisib Combined with the Allosteric MEK Inhibitor Refametinib in Patients with Advanced Cancer.

BackgroundDual inhibition of PI3K and MAPK signaling is conceptually a promising anticancer therapy.ObjectiveThis phase 1b trial investigated the safety, maximum tolerated dose (MTD), recommended phase II dose, pharmacokinetics, tumor response, fluorodeoxyglucose positron emission tomography (FDG-PET) pharmacodynamics, and biomarker explorations for the combination of pan-PI3K inhibitor copanlisib and allosteric MEK inhibitor refametinib in patients with advanced solid tumors.Patients and methodsThis was an adaptive trial with eight dose cohorts combining dose escalation and varying schedules in repeated 28-day cycles. Patients received copanlisib (0.2–0.8 mg/kg intravenously) intermittently (days 1, 8, 15) or weekly (days 1, 8, 15, 22) each cycle, and refametinib (30–50 mg twice daily orally) continuously or 4 days on/3 days off. Patients with KRAS, NRAS, BRAF, or PI3KCA mutations were eligible for the expansion cohort.ResultsIn the dose-escalation (n = 49) and expansion (n = 15) cohorts, the most common treatment-emergent adverse events included diarrhea (59.4%), nausea, acneiform rash, and fatigue (51.6% each). Dose-limiting toxicities included oral mucositis (n = 4), increased alanine aminotransferase/aspartate aminotransferase (n = 3), acneiform rash, hypertension (n = 2 each), and diarrhea (n = 1). MTD was copanlisib 0.4 mg/kg weekly and refametinib 30 mg twice daily. No pharmacokinetic interactions were identified. Decreased tumor FDG uptake and MEK-ERK signaling inhibition were demonstrated during treatment. Best response was stable disease (n = 21); median treatment duration was 6 weeks.ConclusionsDespite sound rationale and demonstrable pharmacodynamic tumor activity in relevant tumor populations, a dose and schedule could not be identified for this drug combination that were both tolerable and offered clear efficacy in the population assessed.Clinicaltrials.gov identifierNCT01392521.Electronic supplementary materialThe online version of this article (10.1007/s11523-020-00714-0) contains supplementary material, which is available to authorized users.

The HDAC and PI3K dual inhibitor CUDC-907 synergistically enhances the antileukemic activity of venetoclax in preclinical models of acute myeloid leukemia.

Venetoclax is a promising agent in the treatment of acute myeloid leukemia (AML), though its antileukemic activity is limited to combination therapies. Mcl-1 downregulation, Bim upregulation, and DNA damage have been identified as potential ways to enhance venetoclax activity. In this study, we combine venetoclax with the dual PI3K and histone deacetylase inhibitor CUDC-907, which can downregulate Mcl-1, upregulate Bim, and induce DNA damage, as well as downregulate c-Myc. We establish that CUDC-907 and venetoclax synergistically induce apoptosis in AML cell lines and primary AML patient samples ex vivo. CUDC-907 downregulates CHK1, Wee1, RRM1, and c-Myc, which were found to play a role in venetoclax-induced apoptosis. Interestingly, we find that venetoclax treatment enhances CUDC-907-induced DNA damage potentially through inhibition of DNA repair. In vivo results show that CUDC-907 enhances venetoclax efficacy in an AML cell line derived xenograft mouse model, supporting the development of CUDC-907 in combination with venetoclax for the treatment of AML.

The PI3K pathway impacts stem gene expression in a set of glioblastoma cell lines

The PI3K pathway controls diverse cellular processes including growth, survival, metabolism, and apoptosis. Nuclear FOXO factors were observed in cancers that harbor constitutively active PI3K pathway output and stem signatures. FOXO1 and FOXO3 were previously published to induce stem genes such as OCT4 in embryonic stem cells. Here, we investigated FOXO-driven stem gene expression in U87MG glioblastoma cells. PI3K-activated cancer cell lines were investigated for changes in gene expression, signal transduction, and clonogenicity under conditions with FOXO3 disruption or exogenous expression. The impact of PI3K pathway inhibition on stem gene expression was examined in a set of glioblastoma cell lines. We found that CRISPR-Cas9-mediated FOXO3 disruption in U87MG cells caused decreased OCT4 and SOX2 gene expression, STAT3 phosphorylation on tyrosine 705 and clonogenicity. FOXO3 over expression led to increased OCT4 in numerous glioblastoma cancer cell lines. Strikingly, treatment of glioblastoma cells with NVP-BEZ235 (a dual inhibitor of PI3K and mTOR), which activates FOXO factors, led to robust increases OCT4 gene expression. Direct FOXO factor recruitment to the OCT4 promoter was detected by chromatin immunoprecipitation analyses using U87MG extracts. We show for the first time that FOXO transcription factors promote stem gene expression glioblastoma cells. Treatment with PI3K inhibitor NVP-BEZ235 led to dramatic increases in stem genes in a set of glioblastoma cell lines. Given that, PI3K inhibitors are actively investigated as targeted cancer therapies, the FOXO-mediated induction of stem genes observed in this study highlights a potential hazard to PI3K inhibition. Understanding the molecular underpinnings of stem signatures in cancer will allow refinements to therapeutic strategies. Targeting FOXO factors to reduce stem cell characteristics in concert with PI3K inhibition may prove therapeutically efficacious.