Mark Douglas

Overcoming resistance to checkpoint blockade therapy by targeting PI3Kγ in myeloid cells

Recent clinical trials using immunotherapy have demonstrated its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies against cytotoxic-T-lymphocyte-associated protein 4 or programmed cell death protein 1/programmed death-ligand 1 have displayed durable clinical responses in various cancers. Although these new immunotherapies have had a notable effect on cancer treatment, multiple mechanisms of immune resistance exist in tumours. Among the key mechanisms, myeloid cells have a major role in limiting effective tumour immunity. Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance. These observations suggest a need for a precision medicine approach in which the design of the immunotherapeutic combination is modified on the basis of the tumour immune landscape to overcome such resistance mechanisms. Here we employ a pre-clinical mouse model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumours. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3Kγ with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumour immune microenvironment and promote cytotoxic-T-cell-mediated tumour regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3Kγ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumours.

Duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocytic leukemia cells to venetoclax (ABT-199)

Duvelisib, an oral dual inhibitor of PI3K-δ and PI3K-γ, is in phase III trials for the treatment of chronic lymphocytic leukemia (CLL) and indolent non-Hodgkin’s lymphoma. In CLL, duvelisib monotherapy is associated with high iwCLL (International Workshop on Chronic Lymphocytic Leukemia) and nodal response rates, but complete remissions are rare. To characterize the molecular effect of duvelisib, we obtained samples from CLL patients on the duvelisib phase I trial. Gene expression studies (RNAseq, Nanostring, Affymetrix array and real-time RT-PCR) demonstrated increased expression of BCL2 along with several BH3-only pro-apoptotic genes. In concert with induction of transcript levels, reverse phase protein arrays and immunoblots confirmed increase at the protein level. The BCL2 inhibitor venetoclax induced greater apoptosis in ex vivo-cultured CLL cells obtained from patients on duvelisib compared with pre-treatment CLL cells from the same patients. In vitro combination of duvelisib and venetoclax resulted in enhanced apoptosis even in CLL cells cultured under conditions that simulate the tumor microenvironment. These data provide a mechanistic rationale for testing the combination of duvelisib and venetoclax in the clinic. Such combination regimen (NCT02640833) is being evaluated for patients with B-cell malignancies including CLL.

The antiproliferative activity of the heat shock protein 90 inhibitor IPI-504 is not dependent on NAD(P)H:quinone oxidoreductase 1 activity in vivo

IPI-504, a water-soluble ansamycin analogue currently being investigated in clinical trials, is a potent inhibitor of the protein chaperone heat shock protein 90 (Hsp90). Inhibition of Hsp90 by IPI-504 triggers the degradation of important oncogenic client proteins. In cells, the free base of IPI-504 hydroquinone exists in a dynamic redox equilibrium with its corresponding quinone (17-AAG); the hydroquinone form binding 50 times more tightly to Hsp90. It has been proposed recently that the NAD(P)H:quinone oxidoreductase NQO1 can produce the active hydroquinone and could be essential for the activity of IPI-504. Here, we have devised a method to directly measure the intracellular ratio of hydroquinone to quinone (HQ/Q) and have applied this measurement to correlate NQO1 enzyme abundance with HQ/Q ratio and cellular activity of IPI-504 in 30 cancer cell lines. Interestingly, the intracellular HQ/Q ratio was correlated with NQO1 levels only in a subset of cell lines and overall was poorly correlated with the growth inhibitory activity of IPI-504. Although artificial overexpression of NQO1 is able to increase the level of hydroquinone and cell sensitivity to IPI-504, it has little effect on the activity of 17-amino-17-demethoxy-geldanamycin, the major active metabolite of IPI-504. This finding could provide an explanation for the biological activity of IPI-504 in xenograft models of cell lines that are not sensitive to IPI-504 in vitro. Our results suggest that NQO1 activity is not a determinant of IPI-504 activity in vivo and, therefore, unlikely to become an important resistance mechanism to IPI-504 in the clinic. [Mol Cancer Ther 2009;8(12):3369–78]

Duvelisib, a novel oral dual inhibitor of PI3K-d,g, is clinically active in advanced hematologic malignancies

Duvelisib is an oral dual inhibitor of phosphoinositide 3-kinase-δ (PI3K-δ) and PI3K-γ in late-stage clinical development for hematologic malignancy treatment. This phase 1 study evaluated maximum tolerated dose (MTD), pharmacokinetics, pharmacodynamics (PD), efficacy, and safety of duvelisib in 210 patients with advanced hematologic malignancies. In the dose escalation phase (n = 31), duvelisib 8 to 100 mg twice daily was administered, with MTD determined as 75 mg twice daily. In the expansion phase (n = 179), patients with indolent non-Hodgkin lymphoma (iNHL), chronic lymphocytic leukemia (CLL), or T-cell lymphoma (TCL) were treated with 25 or 75 mg duvelisib twice daily continuously. Single-dose duvelisib was rapidly absorbed (time to maximum concentration, 1-2 hours), with a half-life of 5.2 to 10.9 hours. PD results showed inhibition of phospho-AKT (S473) in CLL tumor cells following a single dose and near-complete inhibition of CLL proliferation (Ki-67) by cycle 2. Clinical responses were seen across a range of doses and disease subtypes: iNHL overall response rate, 58% (n = 31) with 6 complete responses (CRs); relapsed/refractory CLL, 56% (n = 55) with 1 CR; peripheral TCL, 50% (n = 16) with 3 CR; and cutaneous TCL, 32% (n = 19). Median time to response was ∼1.8 months. Severe (grade ≥3) adverse events occurred in 84% of patients: neutropenia (32%), alanine transaminase increase (20%), aspartate transaminase increase (15%), anemia and thrombocytopenia (each 14%), diarrhea (11%), and pneumonia (10%). These data support further investigation of duvelisib in phase 2 and 3 studies. This trial was registered at clinicaltrials.gov as #NCT01476657.

Activity of the PI3K-δ,g inhibitor duvelisib in a phase 1 trial and preclinical models of T-cell lymphoma

Duvelisib (IPI-145) is an oral inhibitor of phosphatidylinositol 3-kinase (PI3K)-δ/γ isoforms currently in clinical development. PI3K-δ/γ inhibition may directly inhibit malignant T-cell growth, making duvelisib a promising candidate for patients with peripheral (PTCL) or cutaneous (CTCL) T-cell lymphoma. Inhibition of either isoform may also contribute to clinical responses by modulating nonmalignant immune cells. We investigated these dual effects in a TCL cohort from a phase 1, open-label study of duvelisib in patients with relapsed or refractory PTCL (n = 16) and CTCL (n = 19), along with in vitro and in vivo models of TCL. The overall response rates in patients with PTCL and CTCL were 50.0% and 31.6%, respectively (P = .32). There were 3 complete responses, all among patients with PTCL. Activity was seen across a wide spectrum of subtypes. The most frequently observed grade 3 and 4 adverse events were transaminase increases (40% alanine aminotransferase, 17% aspartate aminotransferase), maculopapular rash (17%), and neutropenia (17%). Responders and nonresponders had markedly different changes in serum cytokine profiles induced by duvelisib. In vitro, duvelisib potently killed 3 of 4 TCL lines with constitutive phospho-AKT (pAKT) vs 0 of 7 lines lacking pAKT (P = .024) and exceeded cell killing by the PI3K-δ-specific inhibitor idelalisib. Administration of duvelisib to mice engrafted with a PTCL patient-derived xenograft resulted in a shift among tumor-associated macrophages from the immunosuppressive M2-like phenotype to the inflammatory M1-like phenotype. In summary, duvelisib demonstrated promising clinical activity and an acceptable safety profile in relapsed/refractory TCL, as well as preclinical evidence of both tumor cell-autonomous and immune-mediated effects. This trial was registered at www.clinicaltrials.gov as #NCT01476657.

Pharmaceutical development of IPI‐504, an Hsp90 inhibitor and clinical candidate for the treatment of cancer

IPI‐504 (retaspimycin hydrochloride) is an Hsp90 inhibitor that is the subject of multiple clinical trials for the treatment of cancer. IPI‐504 is an aqueous soluble (>200 mg/ml) hydroquinone hydrochloride salt of 17‐(allylamino)‐17‐demethoxygeldanamycin (17‐AAG), a quinone derivative also undergoing clinical evaluation, albeit with suboptimal formulations that address its inferior aqueous solubility (∼50 µg/ml). IPI‐504 interconverts with 17‐AAG in vivo through oxidation‐reduction reactions that result in a dynamic redox equilibrium. The development challenges associated with redox active molecules are significant due to the pH, oxygen, and temperature sensitivities associated with such chemotypes. The API and sterile drug product manufacturing processes thus warrant the monitoring and control of these key variables. Furthermore, the pharmaceutical development challenges associated with cancer agents that are often fast‐tracked due to unmet medical needs mandate a rapid development cycle with associated regulatory hurdles. Drug Dev Res 71: 429–438, 2010.

Duvelisib, an oral dual PI3K-δ,γ inhibitor, shows clinical and pharmacodynamic activity in chronic lymphocytic leukemia and small lymphocytic lymphoma in a phase 1 study

Duvelisib (IPI‐145), an oral, dual inhibitor of phosphoinositide‐3‐kinase (PI3K)‐δ and ‐γ, was evaluated in a Phase 1 study in advanced hematologic malignancies, which included expansion cohorts in relapsed/refractory (RR) chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and treatment‐naïve (TN) CLL. Per protocol, TN patients were at least 65 years old or had a del(17p)/TP53 mutation. Duvelisib was administered twice daily (BID) in 28‐day cycles at doses of 8‐75 mg in RR patients (n = 55) and 25 mg in TN patients (n = 18.) Diarrhea was the most common nonhematologic AE (TN 78%, RR 47%); transaminase elevations the most frequent lab‐abnormality AE (TN 33.3%, RR 30.9%); and neutropenia the most common ≥grade 3 AE (RR 44%, TN 33%). The overall response rates were 56.4% for RR patients (1.8% CR, 54.5% PR) and 83.3% for TN patients (all PRs); median response duration was 21.0 months in RR patients but was not reached for TN patients. Based upon phase 1 efficacy, pharmacodynamics, and safety, duvelisib 25 mg BID was selected for further investigation in a phase 3 study in RR CLL/SLL.

Abstract 1891: Pharmacological target validation studies of fatty acid synthase in carcinoma using the potent, selective and orally bioavailable inhibitor IPI-9119.

Fatty acid synthase (FASN) is a key enzyme responsible for fatty acids synthesis de novo in mammals. Overexpression of FASN is common in many cancers including prostate, breast and colon cancer and elevated expression of FASN has been linked with poor prognosis and reduced disease-free survival. Experiments with RNAi and small molecule inhibitors suggest that FASN is a metabolic oncogene with an important role in tumor growth and survival and an appealing target for cancer therapy. However, studies utilizing small molecule FASN inhibitors like orlistat and C75 have been confounded by the lack of potency and selectivity, as well as the poor pharmacological properties of these inhibitors. Herein we report pharmacological target validation studies of FASN using a potent, selective and orally bioavailable FASN inhibitor IPI-9119. Building on previous experience with serine hydrolase inhibitors, a series of novel mechanism-based FASN inhibitors were designed based on a tetrazolone carboxamide scaffold. Like orlistat, these analogs are irreversible inhibitors that specifically target the FASN thioesterase domain. Tetrazolone carboxamide analogs were shown to potently inhibit cellular FASN using an occupancy assay and to completely block de novo palmitate synthesis in HCT-116 colon cancer cells using a 13 C-glucose incorporation assay. Lead optimization of the tetrazolone carboxamide series resulted in the identification of IPI-9119 as a tool for in vivo proof-of-concept studies. IPI-9119 is a potent FASN inhibitor in both biochemical (IC 50 ∼1nM) and cellular occupancy assays (IC 50 ∼10nM), and shows more than 400-fold selectivity against several additional serine hydrolases. Importantly, IPI-9119 is orally bioavailable and has pharmacokinetic (PK) properties suitable for in vivo pharmacology studies. IPI-9119 was tested for growth inhibition in cancer cell lines in vitro and tumor xenograft models in vivo. Unexpectedly, in contrast to the knock-down studies and to data reported for orlistat and C75, IPI-9119 failed to elicit anti-proliferative effects in multiple cancer cell lines in vitro. Similarly, PK/PD experiments demonstrated that a single oral dose of IPI-9119 at 200 mg/kg leads to complete and sustained blockade of FASN in HCT-116 tumor xenografts, but IPI-9119 failed to show any anti-tumor activity when dosed as a single agent at 200 mg/kg BID for 10 days. In summary, we identified IPI-9119 as a potent, selective and orally bioavailable FASN inhibitor. Preliminary target validation studies with IPI-9119 in cancer cell lines and an HCT-116 xenograft model suggest that FASN inhibition alone is not sufficient to affect cancer cell proliferation and tumor growth. Further studies exploring combination treatments with IPI-9119 are warranted. Citation Format: Erin Brophy, James Conley, Patrick O9Hearn, Mark Douglas, Culver Cheung, John Coco, Laura D9Anello, Andrew Wylie, Thomas Tibbitts, Gregg Keaney, Lawrence Chan, Adilah Bahadoor, Dan Snyder, Marta Nevalainen, Alfredo Castro, Vito Palombella, Massimo Loda, Stephane Peluso. Pharmacological target validation studies of fatty acid synthase in carcinoma using the potent, selective and orally bioavailable inhibitor IPI-9119. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1891. doi:10.1158/1538-7445.AM2013-1891