PI3Kδ Inhibitor Research Articles

Efficacy and Safety of Add-on Parsaclisib to Ruxolitinib Therapy in Myelofibrosis Patients With Suboptimal Response to Ruxolitinib: Final Results From a Phase 2 Study

Background: Ruxolitinib (RUX) is a potent JAK1/JAK2 inhibitor and is effective in patients (pts) with myelofibrosis (MF). However, suboptimal or declining responses to RUX occur in a subset of pts, possibly due to persistent PI3K pathway activation with chronic JAK inhibitor therapy. Parsaclisib is a potent and highly selective next-generation PI3Kδ inhibitor. We conducted a phase 2 study (NCT02718300) evaluating add-on parsaclisib to stable doses of RUX, for pts with MF who experienced a suboptimal response to RUX (Yacoub A. HemaSphere 2021;5 Suppl 2:512). Here we report efficacy and safety results from the completed study. Methods: Eligible pts had primary or post-essential thrombocythemia/post-polycythemia vera MF with suboptimal response (palpable spleen >10 cm below left subcostal margin [LSM], or palpable splenomegaly 5-10 cm below LSM and presence of 1 symptom score ≥5 or 2 symptom scores ≥3 each using the Screening Symptom Form) after ≥6 months of RUX (5-25 mg twice daily; RUX stable dose, ≥8 wks). Pts remained on their stable RUX dose and were randomized to receive add-on parsaclisib 10 mg or 20 mg once daily (QD) for 8 wks and the same dose once weekly (QW) thereafter (QD/QW group), or parsaclisib 5 mg or 20 mg QD for 8 wks and 5 mg QD thereafter (all QD group) (Figure). Primary efficacy endpoint was change in spleen volume (SV) from baseline to wk 12 by MRI/CT scan. Key secondary endpoints included change in SV to wk 24, change in total symptom score (MFSAF-TSS, MPN-SAF-TSS), and safety. Results: Overall, 32 pts received parsaclisib QD/QW and 42 pts received parsaclisib all QD. Baseline median age was 68.0 (range 41.0-89.0) years and 47.3% of pts were male. The study has been completed. Median treatment duration was 336.5 days; median average daily dose was 5.0 mg/day for parsaclisib and 29.8 mg/day for RUX. Among all pts, 16 (21.6%) were rolled over to continue therapy in an open-label parsaclisib study after ending treatment in the present study. Other common reasons for pts discontinuing treatment were adverse events (13 [17.6%]) and progressive disease (10 [13.5%]); remainder discontinued for physician decision, pt decision, or "Other." Baseline median SV (cm3) was 2414.5 in QD/QW (n=29) and 1877.5 in QD (n=37); median MFSAF-TSS was 10.8 (n=28) and 16.3 (n=32) and median MPN-SAF-TSS was 25.5 (n=28) and 30.0 (n=37), respectively. Median percentage change in SV at wk 12 was −1.6 (n=28) in QD/QW and −15.4 (n=37) in QD, and at wk 24 was −2.5 (n=20) and −19.3 (n=29), respectively. Median percentage change in MFSAF-TSS at wk 12 was −14.0 (n=21) in QD/QW and −32.8 (n=24) in QD, and at wk 24 was −10.0 (n=16) and −44.4 (n=18), respectively. Median percentage change in MPN-SAF-TSS at wk 12 was −19.2 (n=20) in QD/QW and −39.8 (n=30) in QD, and at wk 24 was −43.3 (n=15) and −60.8 (n=26), respectively. Responder analysis for these 3 efficacy variables is shown in the Table. Nonhematologic treatment-emergent adverse events (TEAEs) were primarily grade 1/2. Grade 3/4 nonhematologic TEAEs occurring in >1 pt overall were pneumonia (n=5; 3 QD/QW, 2 QD), fatigue, hypoxia (each n=2; each 1 QD/QW, 1 QD), dyspnea (n=2, all QD), fall, alanine aminotransferase (ALT) increased, aspartate aminotransferase (AST) increased, hypocalcemia (each n=2; all QD/QW). New-onset grade 3 thrombocytopenia was observed in 6/32 pts (18.8%) in QD/QW and 11/42 pts (26.2%) in QD; grade 4 thrombocytopenia was observed in 6/32 pts (18.8%) in QD/QW and 3/42 pts (7.1%) in QD. TEAEs of special interest included grade ≥3 ALT and AST (each n=2; all QD/QW), grade ≥2 diarrhea (n=4) and rash (n=1) (all QD/QW), and any-grade herpes simplex (n= 4; 1 QD/QW, 3 QD) and VZV infection (n= 3; 1 QD/QW, 2, QD). No colitis was reported. TEAEs led to parsaclisib interruption in 16/32 pts in QD/QW and 22/42 pts in QD, and RUX interruption in 5/32 and 8/42 pts, respectively. TEAEs led to parsaclisib discontinuation in 5/32 pts in QD/QW and 4/42 pts in QD, and RUX discontinuation in 2/32 and 2/42 pts, respectively. Conclusion: Final results from the phase 2 study demonstrate improvement in symptoms and SV with add-on parsaclisib in patients with MF having a suboptimal response to ruxolitinib. All daily dosing regimens were more efficacious than daily/weekly dosing. Combination therapy was associated with limited grade 3/4 AEs and TEAE-related discontinuations. Phase 3 studies using 5 mg QD parsaclisib together with ruxolitinib in naive MF patients and ruxolitinib-experienced patients are in progress. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pharmacological Modulation of CD21 Antigen Density Enhances Chimeric Antigen Receptor (CAR-T) Cell Function

Introduction Antigen density has emerged as an important factor regulating CAR-T cell activity with low antigen density being associated with reduced potency in pre-clinical models across several targets. In clinical trials of CD22 CAR-T cells, the low and heterogeneous CD22 expression in B-ALL enabled CD22lo blasts to escape CAR-T cell detection leading to antigen dim relapses (Nat Med. 2018, 24:20-28). We have previously reported on the development of novel CD21 directed CAR-T cells (CAR21) for the treatment of T-cell acute lymphoblastic leukemia (T-ALL) (Blood. 2021, 138:902). Donor derived CAR21 T cells targeting membrane proximal epitopes and utilizing a Fab-CAR architecture were capable of specific cytotoxicity and cytokine secretion against CD21 positive target cells in vitro and were active in vivo. However, in co-culture assays, CAR21 cells secreted less IL-2 against cell lines and primary tumors expressing CD21 at very low antigen density (<1000 copies/cell). We have shown that CD21 is expressed in ~60% of primary T-ALL samples (n=58) although at low antigen density (mean 1262 copies/cell) compared to more established targets such as CD19. We wanted to explore the impact of this low antigen density on CD21 CAR-T function and whether it was possible to upregulate CD21 density by pharmacological means. Results We have previously shown that CD21 is a direct transcriptional target of the NOTCH-MYC pathway and that PI3K/mTOR inhibition of T-ALL results in activation of this pathway and consequent CD21 up-regulation (Leukaemia. 2013, 27:650). We therefore incubated six T-ALL cell lines with four inhibitors of the PI3K/mTOR/AKT pathway (AZD5363, GDC0941, BEZ235 and rapamycin) for 48 and 72hrs and showed surface CD21 up-regulation by flow cytometry of > two-fold at both time points for all inhibitors, with BEZ235 showing the greatest fold increases across all cell lines. Incubation with the gamma-secretase inhibitor L685 resulted in significantly down regulated CD21 expression across all cell lines in keeping with the known NOTCH regulation of CD21 expression. We were next interested in the persistence of this enhanced CD21 expression after drug withdrawal. In two T-ALL cell lines, PF382 and RPMI8402, the impact of BEZ235, rapamycin and L685 was sustained at 48hrs after washing off. PI3K/ mTOR mediated CD21 manipulation did not occur in a CD21 negative T-ALL cell line, normal donor B and T cells or B-ALL patient samples suggesting the effect was specific to T-ALL. When clinically approved PI3K inhibitors (Idelalisib, Duvelisib, Copanlisib, Everolimus) were used, an increase in CD21 expression was again seen across 5 T-ALL cell lines tested. Greatest fold increases (> 2-fold) in CD21 expression were seen with the dual PI3Kα and PI3Kδ inhibitor copanlisib and the mTOR inhibitor rapamycin. No consistent up regulation of CD7 or CCR9 by flow was seen after 48-hour copanlisib incubation suggesting that the effect was specific to CD21. PI3K inhibition was additionally able to mediate 2-fold increases in CD21 expression in a CD21 low patient-derived xenograft T-ALL sample. Further, in a MOLT-4 murine xenograft model of T-ALL, 2 doses of copanlisib were able to produce a 2-fold increase in CD21 expression. These PI3K-mediated increases in CD21 antigen density translated into improved in vitro CAR21 functionality. Across 3 T-ALL cell lines - MOLT-4, CUTLL-1 and RPMI-8402, 48hr pre-incubation with both rapamycin and copanlisib prior to co-culture with CAR21 or CAR19 cells, resulted in improved CD21 specific cytotoxicity, IFN-G and IL-2 release. However, in the same co-culture assay, when copanlisib was added directly to the co-culture rather than via pre-incubation, there was a significant attenuation of IFN-G and IL-2 production by CAR21 suggesting a direct inhibitory effect on CAR-T effector functions in keeping with the known immunosuppressive effects of PI3K inhibitors. Conclusion In summary, we have shown that CD21 expression in T-ALL can be increased through PI3K pathway inhibition and that enhanced CD21 antigen density through pharmacological priming results in improved in vitro CAR21 efficacy. In vivo models analyzing the impact of copanlisib tumor priming on CAR-T efficacy and persistence are ongoing. Target antigen modulation is a promising strategy for enhancing CAR efficacy and reducing risk of antigen negative relapse in low antigen density targets.

Efficacy and Safety of Single-Agent Zandelisib Administered By Intermittent Dosing in Patients with Relapsed or Refractory (R/R) Follicular Lymphoma (FL): Final Results of the Tidal Phase 2 Study

Introduction: Zandelisib is a novel, highly selective, structurally differentiated, oral PI3Kδ inhibitor. Its on-target residence time (≥5 hours) and high volume of distribution lead to higher tumor exposure relative to plasma over time. In a dose optimization Phase 1b study in various B-cell malignancies, zandelisib was evaluated initially by continuous dosing (CD), and then by intermittent dosing (ID) to allow regulatory T-cell repopulation, thereby reducing immune-related adverse events (AEs) (Pagel et al. Lancet Oncol 2022). The TIDAL trial (NCT03768505) started as a randomized, double-blind, 2-arm study to evaluate the safety and efficacy of zandelisib CD (n=16) vs. ID (n=121) in R/R FL and marginal zone lymphoma (MZL) patients (pts). Here we present results from the final analysis of the FL cohort in the ID arm. Methods: Eligible pts ≥18 years with FL Grade I-IIIA, ECOG performance status 0-1, progressive disease after ≥2 prior therapies including an anti-CD20 antibody and chemotherapy, and no prior PI3K inhibitor, provided consent and subsequently received zandelisib 60 mg orally once daily for two 28-day cycles, then ID (days 1-7 of 28-day cycles). The study arm evaluating zandelisib 60 mg daily CD closed early to enrollment after maturing data from the Ph 1b study showed pts on ID having fewer discontinuations due to treatment-emergent AEs and a high rate of durable responses. Study treatment was administered until disease progression or intolerance. The primary efficacy endpoint was objective response rate (ORR) as assessed by blinded independent central review (BICR) using modified Lugano Classification. Secondary endpoints were duration of response (DOR), complete response (CR) rate, median progression-free survival (mPFS), safety and tolerability. Prophylaxis for Pneumocystis jirovecii pneumonia was not mandatory in this single agent trial. Results: Between June 2019 and September 2021, 121 FL pts were enrolled in the ID arm, intent-to-treat (ITT) population, with the initial 91 pts representing the primary efficacy population (PEP). The median number of prior therapies was 3 (range 2-8), 28 pts (23.1%) had prior stem cell transplant, 54 pts (44.6%) were refractory to last therapy, 42 pts (34.7%) had bulky disease (≥5 cm), and 68 pts (56.2%) were POD24. Efficacy data overall and by subgroup (POD24, prior therapy, refractoriness) are shown in the Table. The ORR (CR rate) in the PEP and ITT were 72.5% (36.3%) and 70.2% (34.7%). With median follow-up of 11.3 months in PEP and 9.3 months in ITT, median DOR was estimated at 16.4 and 16.4 months, respectively. Preliminary PFS in the ITT population is shown in the Figure. With a median follow-up of 14.3 months (range: 1.0-30.5) among 121 pts, the most common AEs (all grade/Gr ≥3) were diarrhea (37.2%/5.8%), neutropenia (28.9%/21.5%), nausea (22.3%/0), fatigue (19.0%/0), pyrexia (19.0%/1.7%), and abdominal pain (15.7%/0.8%). 15 pts (12.4%) discontinued therapy due to treatment-related AE. Gr ≥3 AEs of special interest (AESI) in the ID arm were diarrhea in 7 pts (5.8%), lung infection/pneumonia in 6 (5.0%), noninfectious colitis in 4 (3.3%), rash in 4 (3.3 %), stomatitis in 3 (2.5%), and 1 (0.8%) each for ALT and AST increased and noninfectious pneumonitis. In 16 pts in the CD arm, 5 (31.3%) discontinued therapy due to drug-related AE, and 4 (25%) had Gr ≥3 diarrhea/colitis. Gr 5 AEs in the ID arm, excluding COVID-19, were tumor lysis syndrome (1 pt) and pneumonia (1 pt). There were 17 pts with COVID events, fatal in 5. Conclusions: Single agent zandelisib on ID was associated with high rate of durable responses (72.5% ORR, 36.3% CR rate in PEP) in heavily pretreated R/R FL pts, including those with POD24 and refractory disease, and was associated with a relatively lower incidence of Gr ≥3 AESI and AE-related discontinuations, compared with CD. Based on the safety and efficacy profiles observed in this study, these results support further evaluation of zandelisib ID, alone or in combinations, in various B-cell malignancies. Zandelisib plus rituximab vs. chemoimmunotherapy is being evaluated in the randomized phase 3 study COASTAL in R/R FL and MZL (NCT04745832). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A ROR1 Small Molecule Inhibitor (KAN0441571C) Induced Significant Apoptosis of Mantle Cell Lymphoma (MCL) Cells.

The receptor tyrosine kinase orphan receptor 1 (ROR1) is absent in most normal adult tissues but overexpressed in various malignancies and is of importance for tumor cell survival, proliferation, and metastasis. In this study, we evaluated the apoptotic effects of a novel small molecule inhibitor of ROR1 (KAN0441571C) as well as venetoclax (BCL-2 inhibitor), bendamustine, idelalisib (PI3Kδ inhibitor), everolimus (mTOR inhibitor), and ibrutinib (BTK inhibitor) alone or in combination in human MCL primary cells and cell lines. ROR1 expression was evaluated by flow cytometry and Western blot (WB). Cytotoxicity was analyzed by MTT and apoptosis by Annexin V/PI staining as well as signaling and apoptotic proteins (WB). ROR1 was expressed both in patient-derived MCL cells and human MCL cell lines. KAN0441571C alone induced significant time- and dose-dependent apoptosis of MCL cells. Apoptosis was accompanied by decreased expression of MCL-1 and BCL-2 and cleavage of PARP and caspase 3. ROR1 was dephosphorylated as well as ROR1-associated signaling pathway molecules, including the non-canonical WNT signaling pathway (PI3Kδ/AKT/mTOR). The combination of KAN0441571C and ibrutinib, venetoclax, idelalisib, everolimus, or bendamustine had a synergistic apoptotic effect and significantly prevented phosphorylation of ROR1-associated signaling molecules as compared to KAN0441571C alone. Our results suggest that targeting ROR1 by a small molecule inhibitor, KAN0441571C, should be further evaluated particularly in combination with other targeting drugs as a new therapeutic approach for MCL.

223 (PB103) - Non-ATP competitive inhibition of PI3Kδ with IOA-244 shows anti-lymphoma activity

Background: Activation of PI3 K signaling, mainly mediated via PI3Kδ,is a fundamental signaling cascade in lymphomas (Tarantelli et al, 2021). IOA-244 (IOA) is a highly selective and potent PI3Kδ inhibitor with atypical non-ATP competitive activity (Johnson et al, 2019) compared to FDA-approved PI3 K inhibitors idelalisib, copanlisib and duvelisib. IOA is in phase 1 as an immunomodulatory agent for patients with melanoma, mesothelioma, non-small cell lung cancer, myelofibrosis, and follicular and peripheral T cell lymphoma (TCL) as a single agent or in combination (NCT04328844).

IBCL-271 Primary Efficacy and Safety Analysis of a Global Phase II Study of Zandelisib Administered by Intermittent Dosing (ID) in Patients With Relapsed or Refractory (R/R) Follicular Lymphoma (FL): The TIDAL Study

Zandelisib is a selective PI3Kδ inhibitor administered orally at 60 mg once daily (QD) for 2 cycles (response induction), then intermittent dosing (ID) on days 1-7 of subsequent 28-day cycles for maintenance, while potentially enabling regulatory T-cell recovery to reduce risk of immune adverse events (irAEs) seen with continuous PI3Kδ inhibition. In a phase Ib study of zandelisib in 37 R/R FL patients, the overall response rate (ORR) was 87% (78% single agent; 95% with rituximab), with only 8% discontinuations due to irAEs (Pagel ICML 2021). Topline results from the fully enrolled FL population from TIDAL, a global phase II study evaluating zandelisib in R/R indolent lymphomas (NCT03768505). Age ≥18y with FL Grade I-IIIA, progressive disease after ≥2 prior therapies, and no prior PI3K inhibitor. Consent required. FL sample size (planned): 120 patients. Primary efficacy population (PEP): first 91 patients treated. Zandelisib 60 mg QD for 2 cycles, followed by ID during cycle 3+. IRC-assessed ORR (Lugano criteria) after a minimum 6-month follow-up. 91 FL patients in PEP (of 121 enrolled): median 3 prior therapies (range, 2-8), 21 (23%) prior stem cell transplant, 42 (46%) refractory to last therapy, 31 (34%) tumors ≥5 cm, 51 (56%) POD24. ORR was 70.3% (64/91; 95% CI: 59.8%, 79.5%) and complete response (CR) rate was 35.2% (32/91; 95% CI: 25.4%, 45.9%). Responses occurred early: 87.5% (56/91) occurred at end of Cycle 2, 75% (24/91) of CRs at end of Cycle 4. Data still immature for accurate duration of response (DOR) estimation. With median follow-up of 9.4 months (range, 0.8-24) for all 121 patients, 12 (9.9%) discontinued due to any treatment-related AE. Grade 3 AEs of special interest (AESI) included diarrhea (6/121; 5%), colitis (2/121; 1.7%), rash (4/121; 3.3%), stomatitis (3/121; 2.5%), and AST and ALT elevation and non-infectious pneumonitis (1/121 each; 0.8%). Most Grade 3 AESIs (15 [83%]) occurred during daily dosing (cycles 1-3). Zandelisib on ID led to high ORR and CR rates in heavily pretreated FL patients and was associated with a low rate of grade 3 AESI and discontinuations due to treatment-related AEs.

Cutaneous kinase activity correlates with treatment outcomes following PI3K delta inhibition in mice with experimental pemphigoid diseases.

Chronic blistering at the skin and/or mucous membranes, accompanied by a varying degree of inflammation, is the clinical hallmark of pemphigoid diseases that impose a major medical burden. Pemphigoid diseases are caused by autoantibodies targeting structural proteins of the epithelial basement membrane. One major pathogenic pathway of skin blistering and inflammation is activation of myeloid cells following Fc gamma receptor-dependent binding to the skin-bound immune complexes. This process requires activation of specific kinases, such as PI3Kδ, which have emerged as potential targets for the treatment of pemphigoid diseases. Yet, it is unknown if global cutaneous kinase activity present in lesional pemphigoid disease correlates with therapeutic effects following treatment with a given target-selective kinase inhibitor. To address this, we here first determined the kinase activity in three different mouse models of pemphigoid diseases: Antibody transfer-induced mucous membrane pemphigoid (MMP), antibody transfer-induced epidermolysis bullosa acquisita (EBA) and immunization-induced EBA. Interestingly, the kinome signatures were different among the three models. More specifically, PI3Kδ was within the kinome activation network of antibody transfer-induced MMP and immunization-induced EBA, but not in antibody transfer-induced EBA. Next, the therapeutic impact of the PI3Kδ-selective inhibitor parsaclisib was evaluated in the three model systems. In line with the kinome signatures, parsaclisib had therapeutic effects in antibody transfer-induced MMP and immunization-induced EBA, but not in autoantibody-induced EBA. In conclusion, kinase activation signatures of inflamed skin, herein exemplified by pemphigoid diseases, correlate with the therapeutic outcomes following kinase inhibition, demonstrated here by the PI3Kδ inhibitor parsaclisib.

Identification of PI3K/HDAC Dual-targeted inhibitors with subtype selectivity as potential therapeutic agents against solid Tumors: Building HDAC6 potency in a Quinazolinone-based PI3Kδ-selective template

Pharmacological inhibition of PI3Kδ for battling solid tumors is relatively unexplored. Given the potential synergism of concurrent PI3Kδ/HDAC6 inhibition, and the drawbacks of pioneering PI3K/HDAC dual inhibitors, we discovered a novel series of dual-targeted inhibitors via building HDAC6 potency in a PI3Kδ-selective template. SAR study culminated in the discovery of compound 59, which exhibited remarkable inhibitory activity against both PI3Kδ (IC50 = 2.3 nM) and HDAC6 (IC50 = 13 nM), along with acceptable subtype specificity. In addition to the attractive anti-proliferative activities, especially against T47D cell line (IC50 = 0.042 μM), 59 treatment dramatically ablated the tumor immune escape-related STAT3 signaling and lowered PD-L1 expression at two-digit nanomolar level, reflecting the immunomodulatory properties. Due to its subtype selectivity, it demonstrated low cytotoxicity against normal cells. This research validated the therapeutic potential of PI3Kδ/HDAC6 dual inhibitors against solid tumors, attributed to their dual roles in anti-proliferation and anticancer immunomodulation.

Discovery of Novel PI3Kδ Inhibitors Based on the p110δ Crystal Structure.

PI3Kδ is a key mediator of B-cell receptor signaling and plays an important role in the pathogenesis of certain hematological malignancies, such as chronic lymphocytic leukemia. Idelalisib, which targets PI3Kδ specifically, is the first approved PI3K inhibitor for cancer therapy. Recently, we carried out virtual screening, cell-based assays, adapta kinase assays, and molecular dynamic analysis to discover novel PI3Kδ inhibitors and identified NSC348884 as a lead PI3Kδ inhibitor. NSC348884 had an excellent docking score, potent PI3Kδ-inhibitory activity, antitumor effects on various cancer cell lines, and a favorable binding mode with the active site of PI3Kδ. Moreover, through the structural modification of NSC348884, we further discovered comp#1, which forms H-bonds with both Val828 and Lys779 in the ATP binding pocket of PI3Kδ, with a more favorable conformation binding to PI3Kδ. In addition, we found that N1, N1, N2-trimethyl-N2-((6-methyl-1H-benzo[d]imidazol-2-yl) methyl) ethane-1,2-diamine might be a potential scaffold structure. Thus, the result of this study provides a far more efficient approach for discovering novel inhibitors targeting PI3Kδ.

Atropisomerism in Drug Discovery: A Medicinal Chemistry Perspective Inspired by Atropisomeric Class I PI3K Inhibitors.

Atropisomerism is a type of axial chirality resulting from hindered rotation about a σ bond that gives rise to nonsuperimposable stereoisomers (termed "atropisomers"). The inversion of chirality of an atropisomeric axis is a time- and temperature-dependent dynamic process occurring by simple bond rotation. For this reason, the rotational energy barrier (ΔErot) and the interconversion rate between an atropisomeric pair of biologically active molecules are important parameters to consider in drug discovery.Many compounds with atropisomeric axes advance into development every year. The vast majority of them have low rotational energy barriers (ΔErot lower than 20 kcal/mol), meaning they are rapidly equilibrating conformers and considered achiral (class 1 atropisomers). Compounds in class 2 (ΔErot = 20 to 30 kcal/mol) can be challenging to develop given that the stereochemical integrity of the atropisomeric axes can be compromised over time. It has been recommended that small molecule drug candidates containing one or more atropisomeric axes with rotational energy barriers greater than 30 kcal/mol (class 3 atropisomers) should be developed as single atropisomers.In medicinal chemistry, a σ bond with restricted rotation is engineered into a bioactive molecule primarily to limit its number of accessible conformations, thereby minimizing entropic and/or enthalpic energy penalties associated with biological target binding. In addition to enhanced pharmacology, potential positive outcomes of introducing atropisomerism include improved physicochemical properties and superior pharmacokinetics/ADME profiles. The application of atropisomerism in medicinal chemistry has become increasingly enabled due to recent advances in synthesis, purification, and analysis, as described in this special issue and recent review articles.Herein, we discuss two case studies from our own work in which restricting rotation about axes of atropisomerism led to significant improvements in pharmacological, physicochemical, and ADME properties for different series of PI3K inhibitors. In the first instance, a restricted axis of rotation was designed to mitigate an acid-mediated hydrolytic degradation pathway observed in a series of PI3Kδ inhibitors. The conformational constraint disrupts conjugation between a quinazolinone and a pyridine, leading to improved chemical stability under acidic conditions. In the second case study, introduction of a restricted axis of rotation between two heteroaromatic systems in a series of PI3Kβ inhibitors generated pairs of atropisomeric compounds with significantly different biological activities. Advanced profiling also demonstrated clear substrate stereospecificity in regard to metabolism by aldehyde oxidase. Gratifyingly, the eutomer (more active atropisomer) shows significantly less susceptibility for oxidative metabolism relative to the distomer (less active atropisomer). The improvements in potency, selectivity, chemical stability, and metabolic stability discussed in this manuscript are all directly related to the concept of atropisomerism.

MTORC2 Is the Major Second Layer Kinase Negatively Regulating FOXO3 Activity.

Forkhead box O (FOXO) proteins are transcription factors involved in cancer and aging and their pharmacological manipulation could be beneficial for the treatment of cancer and healthy aging. FOXO proteins are mainly regulated by post-translational modifications including phosphorylation, acetylation and ubiquitination. As these modifications are reversible, activation and inactivation of FOXO factors is attainable through pharmacological treatment. One major regulatory input of FOXO signaling is mediated by protein kinases. Here, we use specific inhibitors against different kinases including PI3K, mTOR, MEK and ALK, and other receptor tyrosine kinases (RTKs) to determine their effect on FOXO3 activity. While we show that inhibition of PI3K efficiently drives FOXO3 into the cell nucleus, the dual PI3K/mTOR inhibitors dactolisib and PI-103 induce nuclear FOXO translocation more potently than the PI3Kδ inhibitor idelalisib. Furthermore, specific inhibition of mTOR kinase activity affecting both mTORC1 and mTORC2 potently induced nuclear translocation of FOXO3, while rapamycin, which specifically inhibits the mTORC1, failed to affect FOXO3. Interestingly, inhibition of the MAPK pathway had no effect on the localization of FOXO3 and upstream RTK inhibition only weakly induced nuclear FOXO3. We also measured the effect of the test compounds on the phosphorylation status of AKT, FOXO3 and ERK, on FOXO-dependent transcriptional activity and on the subcellular localization of other FOXO isoforms. We conclude that mTORC2 is the most important second layer kinase negatively regulating FOXO activity.

Treg-driven tumour control by PI3Kδ inhibition limits myeloid-derived suppressor cell expansion

BackgroundRecent studies have demonstrated that blocking the PI3Kδ signalling enzyme (by administering a small molecule inhibitor, PI-3065) can potently improve the anti-tumour T-cell response through direct inhibition of Tregs. This treatment also has a negative impact on MDSC numbers but the primary mechanism driving this effect has remained unclear.MethodsThe 4T1 breast cancer mouse model was used in combination with PI-3065 to gain insights into the effect of PI3Kδ inhibition on MDSCs.ResultsPI-3065 treatment resulted in a concomitant reduction in MDSC expansion and tumour size. However, targeting Tregs independent of PI-3065 was also associated with reduced tumour volume and MDSC numbers. Surgical removal of tumours resulted in a rapid and significant decline in MDSC numbers, whilst ex vivo studies using cells from PI-3065-treated mice demonstrated no direct effect of the inhibitor on MDSC activity.ConclusionsOur data suggest that MDSCs are not inhibited directly by PI-3065 treatment but that their reduced recruitment and immunosuppression within the tumour microenvironment is an indirect consequence of PI3Kδ-inhibition-driven tumour control. This indicates that PI3Kδ inhibition drives tumour immunity by breaking down multiple immunosuppressive pathways through both direct mechanisms (on Treg) and indirect mechanisms, secondary to tumour control (on MDSCs).

Mechanistic Investigation of Lysine-Targeted Covalent Inhibition of PI3Kδ via ONIOM QM:QM Computations.

Phosphoinositide 3-kinase (PI3K) enzymes are important drug targets, especially in oncology, and several inhibitors are currently under investigation in clinical trials for the treatment of lymphocytic leukemia, follicular lymphoma, breast, thyroid, colorectal, and lung cancer. Targeted covalent inhibitors hold significant promise for drug discovery research especially for kinases. Targeting the lysine residues attracts attention as a new strategy in designing targeted covalent inhibitors, since the lysine residue provides several advantages over the traditional cysteine residue. Recently, new highly selective covalent inhibitors of PI3Kδ with activated ester warheads, targeting the conserved Lys779 residue, were reported. Based on the observed kinetics, a covalent inhibition mechanism was proposed, but the atomistic details of the reaction are still not understood. Therefore, in the present work, we have conducted quantum chemical ONIOM M06-2X/6-31+G(d,p):PM6 calculations on the active site cluster structure of PI3Kδ to elucidate the microscopic details of the mechanism of the aminolysis reaction between Lys779 and the ester inhibitors. Our calculations clearly discriminate the noncovalent methyl ester inhibitor and the covalent inhibitors with activated phenolic esters. For the representative p-NO2, p-F, p-H, and p-OCH3 phenolic esters, the Gibbs free energy profiles of alternative mechanistic paths through either Asp782 or Asp911 demonstrate the modulatory role of active site aspartate residues. The most plausible path alters depending on the electron-withdrawing/donating nature of the p-substituted phenolate leaving group. Inhibitors with sufficiently strong electron-withdrawing group prefer direct dissociation of the leaving group from the tetrahedral zwitterion intermediate, while the ones with electron-donating group favor the formation of a neutral tetrahedral intermediate prior to the dissociation. The relative Gibbs free energy barriers of p-NO2 < p-F < p-H < p-OCH3 substituted phenyl esters display the same qualitative trend as the experimentally measured kinact/KI values. Our results provide in depth insight into the mechanism, which can pave the way for optimizing the inhibitor efficiency.

Design, Synthesis, and Development of pyrazolo[1,5-a]pyrimidine Derivatives as a Novel Series of Selective PI3Kδ Inhibitors: Part I-Indole Derivatives.

Phosphoinositide 3-kinase δ (PI3Kδ), a member of the class I PI3K family, is an essential signaling biomolecule that regulates the differentiation, proliferation, migration, and survival of immune cells. The overactivity of this protein causes cellular dysfunctions in many human disorders, for example, inflammatory and autoimmune diseases, including asthma or chronic obstructive pulmonary disease (COPD). In this work, we designed and synthesized a new library of small-molecule inhibitors based on indol-4-yl-pyrazolo[1,5-a]pyrimidine with IC50 values in the low nanomolar range and high selectivity against the PI3Kδ isoform. CPL302253 (54), the most potent compound of all the structures obtained, with IC50 = 2.8 nM, is a potential future candidate for clinical development as an inhaled drug to prevent asthma.

Design, Synthesis, and Development of Pyrazolo[1,5-a]pyrimidine Derivatives as a Novel Series of Selective PI3Kδ Inhibitors: Part II-Benzimidazole Derivatives.

Phosphoinositide 3-kinase (PI3K) is the family of lipid kinases participating in vital cellular processes such as cell proliferation, growth, migration, or cytokines production. Due to the high expression of these proteins in many human cells and their involvement in metabolism regulation, normal embryogenesis, or maintaining glucose homeostasis, the inhibition of PI3K (especially the first class which contains four subunits: α, β, γ, δ) is considered to be a promising therapeutic strategy for the treatment of inflammatory and autoimmune diseases such as systemic lupus erythematosus (SLE) or multiple sclerosis. In this work, we synthesized a library of benzimidazole derivatives of pyrazolo[1,5-a]pyrimidine representing a collection of new, potent, active, and selective inhibitors of PI3Kδ, displaying IC50 values ranging from 1.892 to 0.018 μM. Among all compounds obtained, CPL302415 (6) showed the highest activity (IC50 value of 18 nM for PI3Kδ), good selectivity (for PI3Kδ relative to other PI3K isoforms: PI3Kα/δ = 79; PI3Kβ/δ = 1415; PI3Kγ/δ = 939), and promising physicochemical properties. As a lead compound synthesized on a relatively large scale, this structure is considered a potential future candidate for clinical trials in SLE treatment.

Current status of phosphoinotiside-3 kinase inhibitors in blood cancers.

Treatment of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) underwent paradigm shifts, with targeted agents rapidly displacing chemotherapy. Phosphoinotiside-3 kinase (PI3K) is essential for survival and proliferation of neoplastic B cells and has proven a tractable target in NHL, with four agents receiving FDA approval in the last decade. This review summarizes key data and challenges associated with use of PI3K inhibitors in routine practice. Idelalisib and duvelisib are active in CLL and indolent NHL, including in patients with high-risk features. Despite differential targeting of PI3K isoforms, they exhibit comparable efficacy and adverse event profile including autoimmune events (transaminitis, colitis, pneumonitis), mediated by Treg/Th17 imbalance. Although copanlisib, a pan-PI3K inhibitor, is associated with a distinct safety profile (hyperglycemia, hypertension), preclinical studies indicate that umbralisib, a dual inhibitor of PI3Kδ and casein kinase 1ε, may have less effect on Tregs. However, both drugs may still cause immune-mediated toxicities. With close monitoring and management of adverse events, PI3K inhibitors continue to have a role in therapy of R/R CLL and NHL. Strategies to mitigate adverse events and increase efficacy of PI3K inhibitors include time-limited combination approaches, intermittent dosing schedules.

Discovery, Optimization, and Evaluation of Potent and Selective PI3Kδ-γ Dual Inhibitors for the Treatment of B-cell Malignancies.

Nowadays, PI3Kδ-γ dual inhibitors have been approved for the treatment of B-cell malignancies. Dual inhibition of PI3Kδ and PI3Kγ represents a unique therapeutic opportunity and may confer greater benefits than either isoform inhibition alone in the management of hematological malignancies. However, currently available dual inhibitors of PI3Kδ-γ compromise in at least one of several essential properties in terms of potency, selectivity, and pharmacokinetic (PK) profiles. Hence, the main challenge of our optimization campaign was to identify an oral available PI3Kδ-γ dual inhibitor with an optimum balance of potency, selectivity, and PK profiles. The medicinal chemistry efforts culminated in the discovery of compound 58, which exhibited strong potency and high selectivity along with excellent in vivo profiles as demonstrated through PK studies in rats and through pharmacodynamic studies in an SUDHL-6 xenograft model. All the results suggest that compound 58 may be a promising candidate for the treatment of B-cell malignancies.

Neutrophil Survival Signaling During Francisella tularensis Infection.

Neutrophils are the most abundant and shortest-lived leukocytes in humans and tight regulation of neutrophil turnover via constitutive apoptosis is essential for control of infection and resolution of inflammation. Accordingly, aberrant neutrophil turnover is hallmark of many disease states. We have shown in previous work that the intracellular bacterial pathogen Francisella tularensis markedly prolongs human neutrophil lifespan. This is achieved, in part, by changes in neutrophil gene expression. Still unknown is the contribution of major neutrophil pro-survival signaling cascades to this process. The objective of this study was to interrogate the contributions of ERK and p38 MAP kinase, Class I phosphoinositide 3-kinases (PI3K), AKT, and NF-κB to neutrophil survival in our system. We demonstrate that both ERK2 and p38α were activated in F. tularensis-infected neutrophils, but only p38α MAPK was required for delayed apoptosis and the rate of cell death in the absence of infection was unchanged. Apoptosis of both infected and uninfected neutrophils was markedly accelerated by the pan-PI3K inhibitor LY2094002, but AKT phosphorylation was not induced, and neutrophil death was not enhanced by AKT inhibitors. In addition, isoform specific and selective inhibitors revealed a unique role for PI3Kα in neutrophil survival after infection, whereas only simultaneous inhibition of PI3Kα and PI3kδ accelerated death of the uninfected controls. Finally, we show that inhibition of NF-κB triggered rapid death of neutrophil after infection. Thus, we defined roles for p38α, PI3Kα and NF-κB delayed apoptosis of F. tularensis-infected cells and advanced understanding of Class IA PI3K isoform activity in human neutrophil survival.

S208: EFFICACY AND SAFETY OF ZANDELISIB ADMINISTERED BY INTERMITTENT DOSING (ID) IN PATIENTS WITH RELAPSED OR REFRACTORY (R/R) FOLLICULAR LYMPHOMA: PRIMARY ANALYSIS OF THE GLOBAL PHASE 2 STUDY TIDAL

Background: Zandelisib, a PI3Kδ inhibitor with high target-binding affinity, is administered by intermittent dosing (ID) on days 1 to 7 of 28-day cycles to potentially enable regulatory T-cell repopulation and lower the risk of immune-related adverse events (irAEs) associated with continuous PI3Kδ inhibition. In a phase 1b study in 37 patients (pts) with R/R FL, zandelisib administered daily for two 28-day cycles for tumor debulking then on ID achieved an overall response rate (ORR) of 87% (78% as single agent and 95% with rituximab), a median duration of response (DOR) not reached at median follow-up of 16.9 months, and a low rate (<10%) of grade 3 irAEs (Pagel et al. ICML 2021; #113). Aims: We conducted the TIDAL study (NCT03768505) to further evaluate in a global trial the efficacy and safety of zandelisib in larger group of pts with R/R FL and marginal zone lymphoma (MZL). The MZL cohort is still enrolling and not reported here. Methods: Eligible pts ≥18 years with FL Grade I-IIIA, ECOG performance status 0-1, progressive disease after ≥2 prior therapies including an anti-CD20 antibody and chemotherapy, and no prior PI3Kδ inhibitor, provided consent and then received zandelisib 60 mg daily for two 28-day cycles then on ID. Another study arm evaluating zandelisib 60 mg daily continuously was closed to enrollment early and is not reported here. The planned sample size in FL was 120 pts on ID, with the primary efficacy population (PEP) pre-defined as the first 91 pts treated and the safety population consisting of all FL pts treated. The primary efficacy endpoint was ORR as assessed by independent review using the Lugano criteria and analyzed 6 months after completing enrollment in the PEP. Results: 121 FL pts were enrolled. In the PEP (N=91 pts), the median number of prior therapies was 3 (range 2-8), 21 pts (23%) have received prior stem cell transplant, 42 pts (46%) were refractory to last therapy, 31 pts (34%) had tumors ≥5 cm, and 51 pts (56%) were POD24. The ORR was 70.3% (N=64) (95% CI 59.8-79.5%), with 32 pts (35.2%) achieving a complete response (CR). Responses occurred early, with 85.5% (N=56) achieved in the first 2 cycles of therapy and 75% of CRs (N=24) achieved the first 4 cycles. The data are still immature to estimate accurately the DOR. With a median follow-up of 9.4 months (range 0.8-24) in the safety population of 121 pts, 12 pts (9.9%) discontinued therapy due to any drug-related AE. Grade 3 AEs of special interest (AESI) were diarrhea in 6 pts (5%), colitis in 2 (1.7%), cutaneous rash in 4 (3.3%), stomatitis in 3 (2.5%), and 1 (0.8%) each for AST and ALT elevation, and non-infectious pneumonitis. Grade 3 AESIs primarily (15 of 18, 83%) occurred in cycles 1-3, during daily dosing, with only 3 cases reported on ID in Cycles ≥4. Summary/Conclusion: Zandelisib on ID achieved high ORR (70.3%) and CR rate (35.2%) in heavily pretreated R/R FL pts, and was associated with a low rate (<10%) of grade 3 AESI and discontinuations due to AEs, results comparable to the Phase 1b study. Longer follow-up is needed to estimate median DOR. This profile supports evaluation of zandelisib as a single agent and in combination regimens in various B-cell malignancies, both in R/R disease and in earlier lines of therapy. Zandelisib plus rituximab vs chemoimmunotherapy is being evaluated in the phase 3 study COASTAL in R/R FL and MZL (NCT04745832).

P1116: SAFETY, PHARMACOKINETIC (PK), PHARMACODYNAMIC (PD) AND ACTIVITY OF THE HIGHLY SELECTIVE PHOSPHOINOSITIDE 3-KINASE INHIBITOR DELTA (PI3KΔ) INHIBITOR IOA-244 IN PATIENTS WITH FOLLICULAR LYMPHOMA (FL)

Background: IOA-244 is a highly selective inhibitor of PI3Kδ, and has a favourable safety and ADME profile in patients (pts) with solid malignancies. In pts with uveal melanoma, IOA-244 also reduced circulating Tregs counts at the biologically effective dose (BED) range. Aims: As part of the First-in-human (FiH) dose escalation study, pts with FL received IOA-244 at the BED range established in patients with solid malignancies to confirm its favourable safety and PK profile in pts with haematologic malignancies. Methods: IOA-244 was investigated in a two-part FIH study. Part A explored the continuous daily dosing of IOA-244 at 10, 20, 40 and 80 mg in pts with solid malignancies and at 20 mg and 80 mg in pts with FL. Part B consists of expansion cohorts of specific tumour indications, including pts with lymphoma. Primary objective: safety of the anticipated BED, or the recommended phase 2 dose (RP2D). Secondary objectives: PK; PD (e.g., inhibition of CD63 expression on basophils, changes in immune cell subsets in peripheral blood); Lugano-based responses; PFS and OS Results: While Part A for solid malignancies have previously been reported (Di Giacomo et al 2021), we here report for the first time the safety, PK, PD and activity of pts with FL. This part of the study is still ongoing in patients with FL and is anticipated to be completed in 2022. Pts at the first cohort of 20 mg QD daily (4/4; 2 female and 2 males) had no DLT and no dose interruptions. Transient platelet reduction (G3) and AST/ALT elevation (G2) were observed in 1/4 pts, which improved while pt was on therapy. The PK and ADME profile was consistent with the ones observed in pts with solid malignancies. One patient had FL and DLBCL as prior co-primary malignancy with 7 prior lines of therapy. The other 3 pts had 1-3 prior lines of therapy. The best response was SD (1/3 pts) and all pts progressed after completing 2 cycles. LDH was initially elevated in 2/4 pts which subsequently decreased on therapy. 3/4 pts had low Treg counts at baseline. Additional pts are being recruited at the RP2D for solid malignancies, at 80 mg QD. Reference: Di Giacomo et al. Annals of Oncology (2021) 32 (suppl_7): S1428-S1457. 10.1016/annonc/annonc787 Summary/Conclusion: The PK and ADME profile of IOA-244 in pts with FL appears to match that in pts with solid malignancies. Additional pts at the 80 mg QD dose are expected to match the safety observed in pts with solid malignancies. In contrast to other PI3Kd inhibitors, IOA-244 is highly selective and may therefore target Tregs in pts with FL without off-target effects induced by inhibition of other PI3K isoenzymes.