PI3Kδ Inhibitor Research Articles

Design of Novel Phosphatidylinositol 3-Kinase Inhibitors for Non-Hodgkin's Lymphoma: Molecular Docking, Molecular Dynamics, and Density Functional Theory Studies on Gold Nanoparticles.

Non-Hodgkin's lymphomas are a diverse collection of lymphoproliferative cancers that are much less predictable than Hodgkin's lymphomas with a far greater tendency to metastasize to extranodal sites. A quarter of non-Hodgkin's lymphoma cases develop at extranodal sites and the majority of them involve nodal and extranodal sites. The most common subtypes include follicular lymphoma, chronic/small lymphocytic leukaemia, mantel cell lymphoma, and marginal zone lymphoma. Umbralisib is one of the latest PI3Kδ inhibitors in clinical trials for several hematologic cancer indications. In this study, new umbralisib analogues were designed and docked to the active site of PI3Kδ, the main target of the phosphoinositol-3-kinase/Akt/mammalian target of the rapamycin pathway (PI3K/AKT/mTOR). This study resulted in eleven candidates, with strong binding to PI3Kδ with a docking score between -7.66 and -8.42 Kcal/mol. The docking analysis of ligand-receptor interactions between umbralisib analogues bound to PI3K showed that their interactions were mainly controlled by hydrophobic interactions and, to a lesser extent, by hydrogen bonding. In addition, the MM-GBSA binding free energy was calculated. Analogue 306 showed the highest free energy of binding with -52.22 Kcal/mol. To identify the structural changes and the complexes' stability of proposed ligands, molecular dynamic simulation was used. Based on this research finding, the best-designed analogue, analogue 306, formed a stable ligand-protein complex. In addition, pharmacokinetics and toxicity analysis using the QikProp tool demonstrated that analogue 306 had good absorption, distribution, metabolism, and excretion properties. Additionally, it has a promising predicted profile in immune toxicity, carcinogenicity, and cytotoxicity. In addition, analogue 306 had stable interactions with gold nanoparticles that have been studied using density functional theory calculations. The best interaction with gold was observed at the oxygen atom number 5 with -29.42 Kcal/mol. Further in vitro and in vivo investigations are recommended to be carried out to verify the anticancer activity of this analogue.

Activated PI3K delta syndrome 1 mutations cause neutrophilia in zebrafish larvae.

People with activated PI3 kinase delta syndrome 1 (APDS1) suffer from immune deficiency and severe bronchiectasis. APDS1 is caused by dominant activating mutations of the PIK3CD gene that encodes the PI3 kinase delta (PI3Kδ) catalytic subunit. Despite the importance of innate immunity defects in bronchiectasis, there has been limited investigation of neutrophils or macrophages in APDS1 patients or mouse models. Zebrafish embryos provide an ideal system to study neutrophils and macrophages. We used CRISPR-Cas9 and CRISPR-Cpf1, with oligonucleotide-directed homologous repair, to engineer zebrafish equivalents of the two most prevalent human APDS1 disease mutations. These zebrafish pik3cd alleles dominantly caused excessive neutrophilic inflammation in a tail-fin injury model. They also resulted in total body neutrophilia in the absence of any inflammatory stimulus but normal numbers of macrophages. Exposure of zebrafish to the PI3Kδ inhibitor CAL-101 reversed the total body neutrophilia. There was no apparent defect in neutrophil maturation or migration, and tail-fin regeneration was unimpaired. Overall, the finding is of enhanced granulopoeisis, in the absence of notable phenotypic change in neutrophils and macrophages.

An open label trial of nemiralisib, an inhaled PI3 kinase delta inhibitor for the treatment of Activated PI3 kinase Delta Syndrome

Activated PI3Kδ Syndrome (APDS) is a rare inherited inborn error of immunity caused by mutations that constitutively activate the p110 delta isoform of phosphoinositide 3-kinase (PI3Kδ), resulting in recurring pulmonary infections. Currently no licensed therapies are available. Here we report the results of an open-label trial in which five subjects were treated for 12 weeks with nemiralisib, an inhaled inhibitor of PI3Kδ, to determine safety, systemic exposure, together with lung and systemic biomarker profiles (Clinicaltrial.gov: NCT02593539).Induced sputum was captured to measure changes in phospholipids and inflammatory mediators, and blood samples were collected to assess pharmacokinetics of nemiralisib, and systemic biomarkers.Nemiralisib was shown to have an acceptable safety and tolerability profile, with cough being the most common adverse event, and no severe adverse events reported during the study. No meaningful changes in phosphatidylinositol (3,4,5)-trisphosphate (PIP3; the enzyme product of PI3Kδ) or downstream inflammatory markers in induced sputum, were observed following nemiralisib treatment. Similarly, there were no meaningful changes in blood inflammatory markers, or lymphocytes subsets. Systemic levels of nemiralisib were higher in subjects in this study compared to previous observations.While nemiralisib had an acceptable safety profile, there was no convincing evidence of target engagement in the lung following inhaled dosing and no downstream effects observed in either the lung or blood compartments. We speculate that this could be explained by nemiralisib not being retained in the lung for sufficient duration, suggested by the increased systemic exposure, perhaps due to pre-existing structural lung damage.In this study investigating a small number of subjects with APDS, nemiralisib appeared to be safe and well-tolerated. However, data from this study do not support the hypothesis that inhaled treatment with nemiralisib would benefit patients with APDS.

Molecular Insight into Drug Resistance Mechanism Conferred by Aberrant PIK3CD Splice Variant in African American Prostate Cancer.

Targeting PI3Kδ has emerged as a promising therapy for hematologic and non-hematologic malignancies. Previously, we identified an oncogenic splice variant, PIK3CD-S, conferring Idelalisib resistance in African American (AA) prostate cancer (PCa). In the current study, we employed a comprehensive analysis combining molecular biology, biochemistry, histology, in silico simulation, and in vitro functional assays to investigate the PIK3CD-S expression profiles in PCa samples and to elucidate the drug resistance mechanism mediated by PI3Kδ-S (encoded by PIK3CD-S). The immunohistochemistry, RT-PCR, and Western blot assays first confirmed that PI3Kδ-S is highly expressed in AA PCa. Compared with PCa expressing the full-length PI3Kδ-L, PCa expressing PI3Kδ-S exhibits enhanced drug resistance properties, including a higher cell viability, more antiapoptotic and invasive capacities, and constitutively activated PI3K/AKT signaling, in the presence of PI3Kδ/PI3K inhibitors (Idelalisib, Seletalisib, Wortmannin, and Dactolisib). Molecular docking, ATP-competitive assays, and PI3 kinase assays have further indicated a drastically reduced affinity of PI3Kδ inhibitors with PI3Kδ-S vs. PI3Kδ-L, attributed to the lack of core binding residues in the PI3Kδ-S catalytic domain. Additionally, SRSF2 has been identified as a critical splicing factor mediating exon 20 skipping in PIK3CD pre-mRNA. The inhibition of the SRSF2 activity by SRPIN340 successfully sensitizes AA PCa cells to PI3Kδ inhibitors, suggesting a novel therapeutic option for Idelalisib-resistant tumors.

Circulating Th17 T cells at treatment onset predict autoimmune toxicity of PI3Kδ inhibitors

PI3Kδ inhibitors are approved for the therapy of B cell malignancies, but their clinical use has been limited by unpredictable autoimmune toxicity, despite promising efficacy and evidence that toxicity is associated with improved clinical outcomes. Prior phenotypic evaluation by CyTOF has identified increases in activated CD8 T cells with activation of Th17 T cells, as well as decreases in Tregs, particularly in patients with toxicity. Here we sought to further understand the effects of idelalisib and duvelisib in vitro, and demonstrate that both idelalisib and duvelisib can inhibit T cell proliferation as well as Th1 and Treg differentiation in vitro, while promoting Th2 and Th17 differentiation. We further demonstrate directly using intracellular flow cytometry that autoimmune toxicity in patients is associated with higher absolute numbers of CD4 and CD8 T cells with Th17 differentiation in peripheral blood prior to therapy, and that gastrointestinal tissues from patients with active autoimmune complications of PI3Kδ inhibitors show infiltration with Th17+ T cells. These same tissues show depletion of Tregs as compared to CLL patients without toxicity, suggesting that loss of Tregs may be permissive for Th17 activation to lead to autoimmune toxicity. Clinical trials to restore this balance are warranted.

In vitro and in vivo metabolic profiling of PD105, a PI3Kδ inhibitor, using UHPLC-Q-Exactive plus-MS

PD105, a PI3Kδ inhibitor, is a candidate for the treatment of rheumatoid arthritis. This study aims to identify the metabolic profiling in vitro and in vivo by UHPLC-Q-Exactive Plus-MS. The in vitro metabolism of PD105 was studied by mouse liver microsomes and hepatocytes, while the in vivo metabolic profiling was obtained from mouse plasma, urine, and faeces. A total of 20 metabolites were tentatively identified based on accurate mass, fragment pathways, and characteristic fragment ions, including 4 in vitro and 20 in vivo. The proposed metabolic pathways of PD105 showed that there were 18 phase I metabolites and 2 phase II metabolites. The phase I metabolic pathways included oxidation, hydration, desaturation and oxidative dechlorination, while the phase II metabolic reactions were mainly methylation and arginine conjugation. Among them, oxidation was the main metabolic pathway of PD105. The comprehensive metabolic profiling contributed to further elucidation of pharmacological action and mechanism of PD105.

Targeting Proliferation Signals and the Cell Cycle Machinery in Acute Leukemias: Novel Molecules on the Horizon.

Uncontrolled proliferative signals and cell cycle dysregulation due to genomic or functional alterations are important drivers of the expansion of undifferentiated blast cells in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cells. Therefore, they are largely studied as potential therapeutic targets in the field. We here present the most recent advancements in the evaluation of novel compounds targeting cell cycle proteins or oncogenic mechanisms, including those showing an antiproliferative effect in acute leukemia, independently of the identification of a specific target. Several new kinase inhibitors have been synthesized that showed effectiveness in a nanomolar to micromolar concentration range as inhibitors of FLT3 and its mutant forms, a highly attractive therapeutic target due to its driver role in a significant fraction of AML cases. Moreover, we introduce novel molecules functioning as microtubule-depolymerizing or P53-restoring agents, G-quadruplex-stabilizing molecules and CDK2, CHK1, PI3Kδ, STAT5, BRD4 and BRPF1 inhibitors. We here discuss their mechanisms of action, including the downstream intracellular changes induced by in vitro treatment, hematopoietic toxicity, in vivo bio-availability and efficacy in murine xenograft models. The promising activity profile demonstrated by some of these candidates deserves further development towards clinical investigation.

Design, synthesis and in vitro biological evaluation of 2-aminopyridine derivatives as novel PI3Kδ inhibitors for hematological cancer

Phosphoinositide-3-kinase (PI3K) involves in regulation of proliferation, cell cycle, and apoptosis, and is overexpressed in most of human malignant tumors. Therefore, the development of PI3K inhibitors has attracted great interest in tumor treatment. In this study, we designed and synthesized a series of 2-aminopyridine derivatives via a bioisosterism strategy. Among them, compound MR3278 showed superior PI3Kδ inhibitory activity (IC50 = 30 nM), as well as higher inhibitory activity to most of AML cells (e.g., MOLM-16 and Mv-4-11 cells with IC50 values of 2.6 μM and 3.7 μM, respectively) than Idelalisib. Further cell studies indicated that MR3278 could induce G2/M phase arrests and cell apoptosis of Mv-4-11 cells via PI3K dependent pathway in a dose dependent manner. In addition, in silico physicochemical and ADMET evaluation revealed its drug-like properties with satisfactory toxicity profiles. These results indicate that MR3278 can be identified as a promising new lead compound to the current PI3Kδ inhibitor and is worthy of further profiling.

Development and optimization of a continuous flow ester reduction with LiAlH4 in the synthesis of a key intermediate for a PI3Kδ inhibitor (CPL302415)

Highly productive (11.3 g h−1) continuous flow LAH ester reduction in the synthesis of a new PI3Kδ inhibitor (CPL302415). Comparison of the efficiency and selectivity of various reduction methods (catalytic with stoichiometric and flow with batch).

Mass balance, metabolic disposition, and pharmacokinetics of a novel selective inhibitor of PI3Kδ [14C] SHC014748M in healthy Chinese subjects following oral administration.

SHC014748M is a potent, novel selective PI3Kδ isoform inhibitor and is proposed for the treatment of non-Hodgkin lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma. This study investigated the pharmacokinetics, mass balance, metabolism and excretion of SHC014748M in Chinese male subjects following a single oral dose of 150mg (100μCi) [14C] SHC014748M. Six healthy Chinese male subjects administrated an oral suspension of 150mg (100μCi) [14C] SHC014748M and the samples of blood, urine and feces were collected for measuring. Liquid chromatography-tandem mass spectrometry and liquid scintillation counter were utilized to obtain mass balance and the pharmacokinetic data. The median Tmax for [14C]-radioactivity was 1.6 ± 0.5h after the oral administration of [14C] SHC014748M and the mean Cmax was 3863 ± 354ngEq./mL in plasma, while the mean Cmax, t1/2 values and AUC0-∞ values for total radioactivity in whole blood were 2466 ± 518ngEq./mL, 32.2 ± 30.5h and 66,236 ± 44,232h * ng Eq./mL, respectively. Fecal excretion was proposed as the predominant elimination route, accounting for a mean of 90.68 ± 11.38% of the administered dose, whereas the mean urine excretion was 6.00 ± 1.48% within 336h post-dose. The proposed major metabolic pathway of [14C] SHC014748M in the human body were as follows: (I) monooxidation, (II) glucuronide acid conjugation, and (III) monoxide-hydrogenation. SHC014748M was absorbed, metabolized and excreted with unchanged SHC014748M as its main circulating component in plasma following oral administration. In addition, it was speculated that fecal excretion was the principal excretion pathway; meanwhile, monohydroxy, glucuronide conjugation, oxygen, and hydrogenation were the major clearance pathways of SHC014748M through urine and/or feces. The trial registration number: CTR20202505.

Validated LC-MS/MS method for the determination of copanlisib in mouse dried blood spots.

Copanlisib is a dual PI3K-δ inhibitor, used in follicular lymphoma treatment. In this research, we report a validated LC-MS/MS method for quantifying copanlisib from a mouse dried blood spot (DBS). We validated the method in line with the guidelines of the US Food and Drug Administration. The liquid-liquid extraction technique was used to extract copanlisib from the DBS discs. We used an Atlantis dC18 column and isocratic mobile phase for the chromatographic separation of copanlisib and the internal standard (idelalisib). The flow was 0.90 ml/min. Under the optimized chromatographic conditions, the retentions of copanlisib and the internal standard were 0.98 and 0.93 min, respectively. Each injection total run time was 2.50 min. The MS/MS ion transitions monitored were m/z 481.31→128.00 and 416.10→176.10 for copanlisib and the internal standard (IS) idelalisib, respectively. We have used a broad calibration range (1.01-4,797 ng/ml) with a determination coefficient (r2 ) of 0.997. All of the evaluated parameters met the acceptance criteria. Hematocrit did not influence the DBS copanlisib concentrations. We have used the validated method to derive the intravenous pharmacokinetic parameters by quantifying copanlisib in mouse plasma.

A pyridinesulfonamide derivative FD268 suppresses cell proliferation and induces apoptosis via inhibiting PI3K pathway in acute myeloid leukemia.

Aberration of PI3K signaling pathway has been confirmed to be associated with several hematological malignancies including acute myeloid leukemia (AML). FD268, a pyridinesulfonamide derivative characterized by the conjugation of 7-azaindole group, is a newly identified PI3K inhibitor showing high potent enzyme activity at nanomole concentration. In this study, we demonstrated that FD268 dose-dependently inhibits survival of AML cells with the efficacy superior to that of PI-103 (pan-PI3K inhibitor) and CAL-101 (selective PI3Kδ inhibitor) in the tested HL-60, MOLM-16, Mv-4-11, EOL-1 and KG-1 cell lines. Further mechanistic studies focused on HL-60 revealed that FD268 significantly inhibits the PI3K/Akt/mTOR signaling pathway, promotes the activation of pro-apoptotic protein Bad and downregulates the expression of anti-apoptotic protein Mcl-1, thus suppressing the cell proliferation and inducing caspase-3-dependent apoptosis. The bioinformatics analysis of the transcriptome sequencing data also indicated a potential involvement of the PI3K/Akt/mTOR pathway. These studies indicated that FD268 possesses high potent activity toward AML cells via inhibition of PI3K/Akt/mTOR signaling pathway, which sheds some light on the pyridinesulfonamide scaffold for further optimization and investigation.

A randomized, placebo-controlled phase 3 trial of the PI3Kδ inhibitor leniolisib for activated PI3Kδ syndrome

AbstractActivated phosphoinositide 3-kinase delta (PI3Kδ) syndrome (APDS) is an inborn error of immunity with clinical manifestations including infections, lymphoproliferation, autoimmunity, enteropathy, bronchiectasis, increased risk of lymphoma, and early mortality. Hyperactive PI3Kδ signaling causes APDS and is selectively targeted with leniolisib, an oral, small molecule inhibitor of PI3Kδ. Here, 31 patients with APDS aged ≥12 years were enrolled in a global, phase 3, triple-blinded trial and randomized 2:1 to receive 70 mg leniolisib or placebo twice daily for 12 weeks. Coprimary outcomes were differences from baseline in the index lymph node size and the percentage of naïve B cells in peripheral blood, assessed as proxies for immune dysregulation and deficiency. Both primary outcomes were met: the difference in the adjusted mean change (95% confidence interval [CI]) between leniolisib and placebo for lymph node size was −0.25 (−0.38, −0.12; P = .0006; N = 26) and for percentage of naïve B cells, was 37.30 (24.06, 50.54; P = .0002; N = 13). Leniolisib reduced spleen volume compared with placebo (adjusted mean difference in 3-dimensional volume [cm3], −186; 95% CI, −297 to −76.2; P = .0020) and improved key immune cell subsets. Fewer patients receiving leniolisib reported study treatment-related adverse events (AEs; mostly grades 1-2) than those receiving placebo (23.8% vs 30.0%). Overall, leniolisib was well tolerated and significant improvement over placebo was notable in the coprimary endpoints, reducing lymphadenopathy and increasing the percentage of naïve B cells, reflecting a favorable impact on the immune dysregulation and deficiency seen in patients with APDS. This trial was registered at www.clinicaltrials.gov as #NCT02435173.

Rapid Tumor Debulking of Relapsed/Refractory CLL Patients By PI3Kδ Inhibition and Anti-CD20 Monoclonal Antibody Treatment

Introduction Effective multi-targeted drug combinations could rapidly achieve deep remissions in CLL allowing for defined duration treatment. The combination of a novel highly-specific phosphoinositide 3-kinase delta (PI3Kδ) inhibitor and casein kinase 1 epsilon (CSK1ε) inhibitor, umbralisib (UMB), and a glycoengineered chimeric monoclonal antibody (mAb) targeting a unique epitope on CD20, ublituximab (UBL), with the BCL2 inhibitor venetoclax (VEN) may decrease drug resistance, reduce risk of tumor lysis syndrome (TLS) and achieve higher levels of undetectable minimal residual disease (MRD). This triple agent therapy is being evaluated in relapsed or refractory CLL patients (U2-VEN, ClinicalTrials.gov NCT03379051). Patients received oral 800 mg UMB once-daily. For Cycle 1, UBL was given via IV on Day 1 (150 mg), Day 2 (750 mg), Day 8 (900 mg), Day 15 (900 mg), and then on Day 1 in Cycles 2 and 3 (900 mg); for 3 cycles of 28 days, followed by consolidation with UMB and VEN for cycles 4 to 24. Patient (n = 25) blood samples were collected Pre- and Post-treatment on days of UBL administration during cycles 1-3. Our studies focused on UMB and UBL treatment effects on: 1) The decrease in the circulating CLL tumor burden and thus the subsequent risk of TLS from the addition of VEN to the regimen; and 2) CD20 antigen loss on CLL cells, a major mechanism of anti-CD20 mAb resistance. Methods Blood was analyzed by clinical laboratory automated cell counter and then stained with antibodies against CD3, CD5, CD19, and CD20 for flow cytometry analysis. CLL cell counts were calculated from flow cytometry and clinical laboratory results. To quantitate CD20 surface molecules, peripheral blood mononuclear cells were isolated and stained for flow cytometry analysis with the addition of a Molecules of Equivalent Soluble Fluorochrome standard curve. All human specimen collection and usage was conducted with written informed consent after approval of our Institutional Research Subjects Review Board according to the ethical guidelines of the Declaration of Helsinki. Results Average CLL cell counts (47 x 109/L) decreased rapidly after administration of the first dose of UMB and UBL (C1D1 Post, 26 x 109/L), which rebounded the next day (C1D2 Pre, 43 x 109/L) and then decreased after the second treatment (C1D2 Post, 24 x 109/L). By Day 15, the average CLL cell count had dropped to 9 x 109/L, which decreased further to 5 x 109/L by the start of cycle 3 (Fig. 1). Notably, the variation in CLL cell counts (error bars) decreases over treatment time, suggesting that most CLL patients' tumor burdens are decreasing to a similar low level independent of starting CLL cell count. The change in average CLL cell count between Pre and Post measurements is initially very high (45% decrease C1D1) with decreasing effectiveness over treatment time (8% decrease C3D1). This change could be due to the loss of CLL cell membrane CD20 levels leading to mAb resistance. Pre-treatment (C1D1 Pre) average CLL cell membrane CD20 levels (n=18) were 29,000 CD20 molecules/cell. This decreased to 9,000 molecules/cell after the first treatment (C1D1 Post), stabilized at 9,100 molecules/cell on the next day (C1D2 Pre) and then decreased further after the second treatment (C1D2 Post) to 1600 molecules/cell. After day 8 treatment, CD20 levels remained at 1,000 molecules/cell or less until the end of this study (C3D1). Discussion UMB and UBL rapidly decrease circulating CLL counts over three cycles of treatment, reducing most CLL patient tumor burden to similar low levels. This clinical response could facilitate addition of VEN in the 4thcycle of therapy by decreasing the risk of TLS. The rapid loss of surface CD20 from CLL cells to 1,000 CD20 molecules/cell or less by C1D8 Post has the potential to cause acquired resistance to anti-CD20 mAb therapy. This rapid and sustained loss of CD20 levels is consistent with that reported at standard doses for other anti-CD20 mAbs. However, an effect of concomitant UMB on CD20 levels cannot be ruled out. Previous work suggests that lower anti-CD20 mAb doses can prevent these large decreases in CD20 levels and increase UBL efficacy. In conclusion, our initial data show that treatment with a selective PI3Kδ and CSK1ε inhibitor (UMB) combined with an anti-CD20 mAb (UBL) is effective in decreasing CLL cell counts in all patients assessed in the U2-VEN clinical trial. However, treatment efficacy could be limited by large decreases in the CLL surface CD20 levels. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase II Trial of Acalabrutinib in Combination with PI3Kδ Inhibitor Umbralisib and the Anti-CD20 Antibody Ublituximab (AU2) in Patients with Previously Untreated Mantle Cell Lymphoma (MCL)

Background. Chemo-immunotherapy followed by autologous stem cell transplant remains the standard approach in treatment of patients with newly diagnosed MCL but is not curative. Furthermore, the majority of patients are poor candidates for this approach due to age, comorbidities, or high genetic risk. Bruton tyrosine kinase inhibitors have high efficacy and are well tolerated in relapsed MCL, and have already been successfully used in frontline therapy. Here we report initial results of a phase 2 study of AU2 in patients with previously untreated MCL (NCT04783415). Methods. This ongoing open-label phase 2 investigator-sponsored study enrolled patients with previously untreated MCL, aged ≥65 years, those unwilling to undergo intensive induction, and/or patients with high-risk genetics (TP53 aberrations, complex karyotype [CK]). Acalabrutinib was given at 100 mg po twice daily, umbralisib 800 mg po daily (amended to days 1-14 of cycle 1 and days 1-7 of subsequent cycles) starting with cycle 1 day 1; ublituximab 900 mg intravenously on days 1, 8, 15 of cycle 1 and day 1 of subsequent cycles. Each cycle was 28 days. After 6 cycles, patients continued maintenance with oral agents and ublituximab every 2 cycles (planned for 24 cycles). The primary study objective was efficacy (compete response rate) by Lugano criteria; secondary objective was safety. The study was suspended to enrollment by the FDA in February 2022 due to general safety concerns with umbralisib and other PI3K inhibitors. Results. Twelve patients were enrolled. Median age was 70 (55-79) years, 9/12 (75%) were male; all had an ECOG performance status 0-1. 6/12 patients had a TP53 aberration, and one additional patient had CK. The first two patients enrolled on study developed grade 3 and 4 AST/ALT elevation by mid-cycle 2. As a result, the trial was amended to administer umbralisib on days 1-14 of cycle 1 and days 1-7 of subsequent cycles. In total, four patients were unable to tolerate acalabrutinib due to recurrent AST/ALT abnormalities and continued on U2 therapy alone. The remaining eight patients tolerated all three drugs. All 12 patients achieved complete response (ORR 100%, CR 100%). At this time, 10/12 patients remain on therapy. One patient discontinued after 9 cycles (for progressive disease), and another patient discontinued after 4 cycles due to an adverse event (HSV encephalitis). The 10 patients still on treatment have received a median 12.5 (range 6-16) cycles. The most common treatment-related adverse events (AEs, any grade) were infusion-related reactions (Table 1); the most common grade 3+ AEs were ALT/AST elevation and infusion-related reaction. Discussion. Thus, AU2 is a highly effective regimen in patients with previously untreated MCL, including those with high-risk genetics (100% CR rate). While a combination of continuous umbralisib and acalabrutinib was associated with liver function test abnormalities, intermittent dosing of umbralisib was well tolerated. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Combinatorial PI3K/AKT Pathway Inhibition As a Therapeutic Approach in Diffuse Large B-Cell Lymphoma

Background. Diffuse large B-cell lymphoma (DLBCL) is the most frequent type of non-Hodgkin lymphoma (NHL, ~ 25-30% of NHL cases). Currently, DLBCL treatment is based on non-specific chemotherapy combination (cyclophosphamide, doxorubicin, vincristine, and prednisone) with anti-CD20 monoclonal antibody. Although the DLBCL survival dramatically improved during last decades, approximately 40 % of DLBCL patients could not be cured with the currently used therapeutic approaches. Novel approaches are, therefore, needed to improve DLBCL survival and to decrease treatment related toxicity. Since many DLBCL tumors depend on phosphatidylinositol 3-kinase /protein kinase B (PI3K/AKT) signaling pathway, it represents one of the possible novel therapeutic targets. Despite promising results in various cancers, PI3K/AKT pathway inhibitors show only partial efficacy, low specificity, and resistance development in lymphomas. Since the PI3K/AKT activity is highly regulated, we hypothesize that the PI3K/AKT pathway needs to be inhibited at multiple levels simultaneously to achieve its full and lasting inhibition. Methods. To provide a proof of principle of multilevel vertical PI3K/AKT inhibition, we tested the effect of four inhibitors targeting the PI3K/AKT pathway at four different levels: PI3Kδ inhibitor idelalisib, PDPK1 inhibitor GSK2334470, AKT inhibitor ipatasertib, and mTOR inhibitor rapamycin on a spectrum of commonly used DLBCL model cell lines. Precise measurement of AKT activity in living cells was done using our recently published genetically encoded Förster Resonance Energy Transferred (FRET) based AKT activity reporter including novel methodology for flow cytometry-based FRET measurement (PMID: 34128311) allowing medium throughput and measurement in kinetics setting. Results. First, we measured how single inhibitors affect the AKT activity. The AKT activity IC50 concentrations were in 100 nM range except for rapamycin that had no AKT inhibitory effect. These concentrations were approximately 10 times lower in comparison to IC50 values calculated from net cell growth inhibition assessing the overall inhibitor toxicity. This suggests compensatory mechanisms and/or off target effects of PI3K/AKT inhibitors. Precise measurement of AKT activity allowed us to identify the minimal concentration of each inhibitor at which AKT activity was fully inhibited. Kinetics and short time course experiments showed that maximal AKT inhibition is achieved within hours of inhibitors treatment initiation. Importantly, pharmacological inhibition of one or two members of PI3K/AKT signaling cascade did not lead to lasting AKT activity inhibition. Cellular contra-regulatory mechanisms compensated for the decrease of AKT activity within 24 hours even in the presence of the inhibitors. Moreover, we detected a massive increase in AKT activity following release of the cells from media with PI3K/AKT pathway inhibitors. This further illustrates the powerful cellular contra-regulatory mechanisms and highlights the need for combinatorial approach. Combination of at least three inhibitors was necessary to achieve lasting AKT activity decrease (long time course experiment, Fig. 1A). Cross titration experiments further confirmed synergistic effect between tested PI3K/AKT pathway inhibitors on cell growth inhibition and allowed to determine the concentration ratios of their maximal synergism. Importantly, testing of single inhibitors and two, three, or all four inhibitors in fixed concentration combinations identified a substantial decrease in cell growth/toxicity IC50 concentrations of individual inhibitors (Fig. 1B). Conclusions. Concept of inhibiting more members of PI3K/AKT pathway was suggested before using combinations and/or dual inhibitors of AKT and mTOR, however, our data suggest that more than two inhibitors are needed for lasting PI3K/AKT inhibition. Moreover, we provided a proof of principle for further multilevel PI3K/AKT signaling inhibition testing, including in vivo cell line and/or patient derived xenograft models. Our data suggest that proposed combinations should allow significant decrease of inhibitors concentrations limiting off-target effect. This approach might overcome above mentioned issues of PI3K/AKT pathway inhibition and allow full therapeutical utilization of this pathway inhibition possibly applicable to other lymphoma types. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Safety and Efficacy of the PI3Kδ Inhibitor Zandelisib in Combination with the BTK Inhibitor Zanubrutinib in Patients with Relapsed/Refractory (R/R) Follicular Lymphoma (FL) or Mantle Cell Lymphoma (MCL)

Safety and Efficacy of the PI3Kδ Inhibitor Zandelisib in Combination with the BTK Inhibitor Zanubrutinib in Patients with Relapsed/Refractory (R/R) Follicular Lymphoma (FL) or Mantle Cell Lymphoma (MCL)

Dual PI3Kδγ inhibition demonstrates potent anticancer effects in diffuse large B-cell lymphoma models: Discovery and preclinical characterization of LL-00084282

Phosphoinositide 3-kinase (PI3K) pathway mediates key signaling events downstream to B-cell receptor (BCR) for survival of mature B-cells, and overexpression or overactivation of PI3Kδ is crucial for B-cell malignancies such as diffuse large B-cell lymphoma (DLBCL). Small molecule PI3Kδγ inhibitors, with a known potential to reduce activated B-cell (ABC)-DLBCL transformation, form an important class of therapeutics approved for follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL). In this study, we describe discovery of a potent, selective and efficacious dual PI3Kδγ inhibitor, LL-00084282, having a differentiated efficacy profile in human ABC- and germinal center B-cell (GCB)-DLBCL cell lines. LL-00084282 displayed high potency and superior PI3Kδγ engagement with excellent selectivity over other PI3K isoforms at both IC50/90 concentrations in biochemical and cell-based assays. In contrast to selective PI3Kδ inhibitors, LL-00084282 showed superior and potent anticancer activity in both ABC- and GCB-DLBCL cell lines. LL-00084282 demonstrated in-vivo efficacy in OCI-Ly10 and SU-DHL-6 xenografts with good tolerability. Furthermore, LL-00084282 inhibited pro-inflammatory cytokine secretion and reduced basophil activation in human PBMCs, showing potential implications in immunoinflammatory conditions. Good pharmacokinetic properties in higher species and desirable efficacy profile highlights potential of this novel PI3Kδγ inhibitor for further clinical evaluation in DLBCL patients.

Safety and Efficacy of Parsaclisib in Combination with Rituximab, Bendamustine + Rituximab, or Ibrutinib in Patients with Previously Treated B-Cell Lymphoma: Analysis of a Phase 1 Dose-Finding Study (CITADEL-112)

Background: Rituximab (RIT)-based chemoimmunotherapy regimens are backbone treatment for indolent (follicular, marginal zone) and aggressive (diffuse large B-cell, mantle cell) B-cell lymphomas. For relapsed or refractory (R/R) disease, anti-CD20 antibodies can be combined with chemotherapy or targeted therapies, such as inhibitors of Bruton's tyrosine kinase (eg, ibrutinib) and phosphoinositide 3-kinase (PI3K). Parsaclisib is a potent and highly selective next-generation PI3Kδ inhibitor. In a preliminary safety analysis of CITADEL-112 (NCT03424122), an open-label phase 1 study of parsaclisib plus investigator choice standard of care (RIT, RIT + bendamustine [BEN], or ibrutinib [IBR]) in patients (pts) with R/R B-cell lymphoma, manageable tolerability was observed (Sancho JM, et al. HemaSphere 2022;6 Suppl 3:P1102). Here we present updated safety and initial efficacy data from CITADEL-112. Methods: Enrolled pts were ≥18 years and had histologically confirmed DLBCL, FL, MCL, or MZL, ECOG performance status of 0-2, were R/R to ≥1 (≥2 for FL) prior systemic therapy, and ineligible for stem cell transplantation. Pts received parsaclisib 20 mg orally once daily (QD) for 8 weeks then 20 mg once weekly (QW) in combination with either RIT 375 mg/m2 IV QW for 4 doses in cycle 1 (± an additional 4 QW doses) in Cohort A, or RIT 375 mg/m2 IV on day 1 + BEN 90 mg/m2 on day 1 and day 2 of each 28-day cycle for ≤6 cycles in Cohort B, or IBR 560 mg QD in Cohort C. A 3+3 design with dose de-escalation identified the maximum tolerated dose of QD parsaclisib within each treatment regimen independently. Pts received treatment until disease progression, unacceptable toxicity, or withdrawal. The primary endpoint was safety and tolerability. Investigator-determined objective response rate (ORR) and duration of response (DOR) were exploratory endpoints. Results: At data cutoff (January 14, 2022), 50 pts were treated (16 pts each in Cohorts A and C, 18 pts in Cohort B) and 5 pts were ongoing treatment (all in Cohort B). Most pts had received ≥2 prior systemic treatments; the most common disease subtypes were FL in Cohort A and DLBCL in Cohorts B and C (Table 1). Median (range) dose of QD parsaclisib was 20.0 (13.3-20.0), 17.9 (2.8-20.0), and 20.0 (7.1-20.0) mg/day in Cohorts A, B, and C, respectively, and median (range) overall duration (QD+QW) of parsaclisib treatment was 5.1 (0.5-22.9), 9.3 (0.5-33.7), and 9.3 (0.6-37.7) months. The most common reasons for discontinuation were progressive disease (75.0%, 44.4%, and 56.3% in Cohorts A, B, and C, respectively) and adverse events (AEs; 12.5%, 16.7%, and 6.3%, respectively). One pt in Cohort B experienced a dose-limiting toxicity of grade 4 neutropenia for 14 days. All pts experienced ≥1 treatment-emergent AE (TEAE); most common any-grade and grade ≥3 TEAEs are shown in Table 2. Grade ≥3 and serious TEAEs were experienced by 75.0% and 37.5% of pts, respectively, in Cohort A, 88.9% and 33.3% of pts in Cohort B, and 62.5% and 50.0% of pts in Cohort C. Serious TEAEs occurring in >1 pt were COVID-19, diarrhea, and pneumonia (n=2 each) in Cohort A, and atrial fibrillation (n=2) in Cohort C. TEAEs with fatal outcome occurred in 2 pts in Cohort A (COVID-19 and COVID-19 pneumonia [n=1], interstitial lung disease [n=1]) and 1 pt in Cohort C (COVID-19 pneumonia, septic shock, acute kidney injury, respiratory failure). TEAEs leading to discontinuation of parsaclisib occurred in 2 pts in Cohort A (diarrhea, streptococcal pneumonia [n=1 each]), 3 pts in Cohort B (neutropenia, maculopapular rash [n=1 each], diarrhea and renal failure [n=1]), and 1 pt in Cohort C (thrombocytopenia). TEAEs leading to parsaclisib dose interruption or reduction occurred in 75.0% and 18.8% of pts, respectively, in Cohort A, 66.7% and 27.8% of pts in Cohort B, and 56.3% and 18.8% of pts in Cohort C. ORRs (95% confidence interval [CI]) were 81.3% (54.4-96.0), 55.6% (30.8-78.5), and 50.0% (24.7-75.3) for Cohorts A, B, and C, respectively; 18.8%, 33.3%, and 12.5% of pts had a complete metabolic response/complete response, respectively (Table 1). Median (95% CI) DOR was 5.2 months (1.7-15.8) for Cohort A, not reached (12.1-not evaluable [NE]) for Cohort B, and 13.4 months (0.7-NE) for Cohort C. Conclusion: Parsaclisib 20 mg QD for 8 weeks followed by 20 mg QW in combination with RIT, RIT+BEN, or IBR in pts with R/R B-cell lymphomas had a manageable tolerability profile with no unexpected safety concerns, and demonstrated promising efficacy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Interim Analysis of Safety and Hematological Parameters of an Ongoing Long-Term Open-Label Extension Study of Investigational PI3Kδ Inhibitor Leniolisib for Patients with Activated PI3K Delta Syndrome (APDS) through December 2021

Interim Analysis of Safety and Hematological Parameters of an Ongoing Long-Term Open-Label Extension Study of Investigational PI3Kδ Inhibitor Leniolisib for Patients with Activated PI3K Delta Syndrome (APDS) through December 2021