Melissa Pink

Discovery of a potent and orally active hedgehog pathway antagonist (IPI-926).

Recent evidence suggests that blocking aberrant hedgehog pathway signaling may be a promising therapeutic strategy for the treatment of several types of cancer. Cyclopamine, a plant Veratrum alkaloid, is a natural product antagonist of the hedgehog pathway. In a previous report, a seven-membered D-ring semisynthetic analogue of cyclopamine, IPI-269609 (2), was shown to have greater acid stability and better aqueous solubility compared to cyclopamine. Further modifications of the A-ring system generated three series of analogues with improved potency and/or solubility. Lead compounds from each series were characterized in vitro and evaluated in vivo for biological activity and pharmacokinetic properties. These studies led to the discovery of IPI-926 (compound 28), a novel semisynthetic cyclopamine analogue with substantially improved pharmaceutical properties and potency and a favorable pharmacokinetic profile relative to cyclopamine and compound 2. As a result, complete tumor regression was observed in a Hh-dependent medulloblastoma allograft model after daily oral administration of 40 mg/kg of compound 28.

Development of 17-allylamino-17-demethoxygeldanamycin hydroquinone hydrochloride (IPI-504), an anti-cancer agent directed against Hsp90

Heat shock protein 90 (Hsp90) is an emerging therapeutic target of interest for the treatment of cancer. Its role in protein homeostasis and the selective chaperoning of key signaling proteins in cancer survival and proliferation pathways has made it an attractive target of small molecule therapeutic intervention. 17-Allylamino-17-demethoxygeldanamycin (17-AAG), the most studied agent directed against Hsp90, suffers from poor physical-chemical properties that limit its clinical potential. Therefore, there exists a need for novel, patient-friendly Hsp90-directed agents for clinical investigation. IPI-504, the highly soluble hydroquinone hydrochloride derivative of 17-AAG, was synthesized as an Hsp90 inhibitor with favorable pharmaceutical properties. Its biochemical and biological activity was profiled in an Hsp90-binding assay, as well as in cancer-cell assays. Furthermore, the metabolic profile of IPI-504 was compared with that of 17-AAG, a geldanamycin analog currently in clinical trials. The anti-tumor activity of IPI-504 was tested as both a single agent as well as in combination with bortezomib in myeloma cell lines and in vivo xenograft models, and the retention of IPI-504 in tumor tissue was determined. In conclusion, IPI-504, a potent inhibitor of Hsp90, is efficacious in cellular and animal models of myeloma. It is synergistically efficacious with the proteasome inhibitor bortezomib and is preferentially retained in tumor tissues relative to plasma. Importantly, it was observed that IPI-504 interconverts with the known agent 17-AAG in vitro and in vivo via an oxidation-reduction equilibrium, and we demonstrate that IPI-504 is the slightly more potent inhibitor of Hsp90.

PI3Kγ is a molecular switch that controls immune suppression

Macrophages play critical, but opposite, roles in acute and chronic inflammation and cancer. In response to pathogens or injury, inflammatory macrophages express cytokines that stimulate cytotoxic T cells, whereas macrophages in neoplastic and parasitic diseases express anti-inflammatory cytokines that induce immune suppression and may promote resistance to T cell checkpoint inhibitors. Here we show that macrophage PI 3-kinase γ controls a critical switch between immune stimulation and suppression during inflammation and cancer. PI3Kγ signalling through Akt and mTor inhibits NFκB activation while stimulating C/EBPβ activation, thereby inducing a transcriptional program that promotes immune suppression during inflammation and tumour growth. By contrast, selective inactivation of macrophage PI3Kγ stimulates and prolongs NFκB activation and inhibits C/EBPβ activation, thus promoting an immunostimulatory transcriptional program that restores CD8+ T cell activation and cytotoxicity. PI3Kγ synergizes with checkpoint inhibitor therapy to promote tumour regression and increased survival in mouse models of cancer. In addition, PI3Kγ-directed, anti-inflammatory gene expression can predict survival probability in cancer patients. Our work thus demonstrates that therapeutic targeting of intracellular signalling pathways that regulate the switch between macrophage polarization states can control immune suppression in cancer and other disorders.

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.

Discovery of a Selective Phosphoinositide-3-Kinase (PI3K)-γ Inhibitor (IPI-549) as an Immuno-Oncology Clinical Candidate

Optimization of isoquinolinone PI3K inhibitors led to the discovery of a potent inhibitor of PI3K-γ (26 or IPI-549) with >100-fold selectivity over other lipid and protein kinases. IPI-549 demonstrates favorable pharmacokinetic properties and robust inhibition of PI3K-γ mediated neutrophil migration in vivo and is currently in Phase 1 clinical evaluation in subjects with advanced solid tumors.

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]

The phosphoinositide-3 kinase (PI3K)-δ,γ inhibitor, duvelisib shows preclinical synergy with multiple targeted therapies in hematologic malignancies

Duvelisib is an orally active dual inhibitor of PI3K-δ and PI3K-γ in clinical development in hematologic malignancies (HM). To identify novel pairings for duvelisib in HM, it was evaluated alone and in combination with 35 compounds comprising a diverse panel of standard-of-care agents and emerging drugs in development for HM. These compounds were tested in 20 cell lines including diffuse large B-cell, follicular, T-cell, and mantle cell lymphomas, and multiple myeloma. Single agent activity was seen in fourteen cell lines, with a median GI50 of 0.59 μM. A scalar measure of the strength of synergistic drug interactions revealed a synergy hit rate of 19.3% across the matrix of drug combinations and cell lines. Synergy with duvelisib was prominent in lymphoma lines with approved and emerging drugs used to treat HM, including dexamethasone, ibrutinib, and the BCL-2 inhibitor venetoclax. Western blotting revealed that certain duvelisib-treated cell lines showed inhibition of phosphorylated (p) AKT at serine 473 only out to 12 hours, with mTORC2 dependent re-phosphorylation of pAKT evident at 24 hours. Combination with dexamethasone or ibrutinib, however, prevented this reactivation leading to durable inhibition of pAKT. The combination treatments also inhibited downstream signaling effectors pPRAS40 and pS6. The combination of duvelisib with dexamethasone also significantly reduced p-4EBP1, which controls cap dependent translation initiation, leading to decreased levels of c-MYC 6 hours after treatment. In support of the in vitro studies, in vivo xenograft studies revealed that duvelisib in combination with the mTOR inhibitor everolimus led to greater tumor growth inhibition compared to single agent administration. These data provide a rationale for exploring multiple combinations in the clinic and suggest that suppression of mTOR-driven survival signaling may be one important mechanism for combination synergy.

Abstract B032: The PI3K-γ inhibitor, IPI-549, increases antitumor immunity by targeting tumor-associated myeloid cells and remodeling the immune-suppressive tumor microenvironment

The PI3 kinases (PI3K) belong to a family of signal-transducing enzymes that mediate key cellular functions in cancer and immunity. The PI3K-gamma (γ) isoform plays an important role in macrophage/myeloid cell function and migration, and a role for PI3K-γ in tumor growth and immune tolerance has been established in studies utilizing PI3K-γ knockout (KO) mice (Schmid et al., Cancer Cell, 2011; Gunderson et al., Cancer Discovery, 2015). We propose that pharmacological inhibition of PI3K-γ in myeloid cells can alter the tumor-immune microenvironment leading to enhanced antitumor T-cell responses. IPI-549 is an oral, potent, and selective inhibitor of PI3K-γ. Prior studies showed single agent antitumor activity in multiple murine tumor models, and enhanced antitumor activity and improved survival when combined with immune-checkpoint blockade. This antitumor activity is dependent on the presence of both immune-suppressive tumor-associated CD11b+ myeloid cells and CD8+ cytotoxic T cells. IPI-549 can reduce the T-cell-suppressive activity of both murine and human myeloid-derived suppressor cells in vitro (Kutok et al, 2015 CRI-CIMT-EATI-AACR Cancer Immunotherapy Meeting; De Henau et al, 2016 AACR Annual Meeting). We now show that IPI‑549 treatment of tumor‑bearing mice leads to a shift in tumor-associated myeloid cells from an immunosuppressive M2 phenotype to a proinflammatory M1 phenotype, characterized by reduced CD206 expression and enhanced expression of MHC class II and NOS2. Compared to vehicle-treated controls, short-term (9 days) treatment of CT26 tumor‑bearing animals with IPI‑549 revealed an increased frequency of circulating tumor-specific T cells, an increased percentage of tumor-infiltrating CD8+IFNγ+ T cells, and a reduced percentage of CD4+Foxp3+ regulatory T cells, leading to a trend towards increasing the CD8+/T-reg cell ratio. Treatment of 4T1 and B16GM tumor-bearing mice with IPI-549 for 14 days led to a significant increase in the CD8+/T-reg cell ratio. Together these data show that IPI-549 treatment leads to a proinflammatory tumor microenvironment. Importantly, gene and protein expression analysis of whole tumor tissue collected from IPI-549-treated mice revealed a cytotoxic T-cell signature characterized by increased production of proinflammatory cytokines, and enhanced expression costimulatory and coinhibitory genes relative to vehicle-treated animals. These findings indicate that IPI-549 increases antitumor immunity by remodeling the tumor-immune microenvironment via blockade of tumor-associated myeloid cells. In addition, the up-regulation of costimulatory and coinhibitory genes with IPI-549 treatment provides a mechanistic rationale for the observed combination activity with immune checkpoint inhibition. IPI-549 is currently in Phase I development, both as a single agent and in combination with an anti-PD-1 antibody, in solid tumors (ClinicalTrials.gov NCT02637531). Citation Format: Matthew Rausch, Jeremy Tchaicha, Thomas Tibbitts, Olivier De Henau, Sujata Sharma, Melissa Pink, Joseph Gladstone, Jennifer Proctor, Mark Douglas, Howard Stern, Taha Merghoub, Jedd Wolchok, Karen McGovern, Jeff Kutok, David Winkler. The PI3K-γ inhibitor, IPI-549, increases antitumor immunity by targeting tumor-associated myeloid cells and remodeling the immune-suppressive tumor microenvironment [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B032.

Abstract B029: The potent and selective phosphoinositide-3-kinase-gamma inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment

Introduction: The phosphoinositide-3-kinase (PI3K) lipid kinases are a family of kinase isoforms that transduce signals in response to various stimuli in different cell types. The PI3K-γ isoform is expressed in immune cells and has limited, if any, expression in epithelial cancer cells. Genetic deletion and kinase-dead knock-in studies highlight a key role for PI3K-γ in the development and function of myeloid-derived cells that constitute a key component of the immune suppressive tumor microenvironment (Joshi Mol Canc Res 2014; Schmid Canc Cell 2011). Targeting PI3K-γ in these tumor-associated myeloid cells could therefore inhibit the immune suppressive tumor microenvironment, enabling the immune system to attack tumor cells more effectively. To date, potent and selective PI3K-γ inhibitors with drug-like properties have not been available to test this hypothesis. We now report the structure, biochemical, cellular, and in vivo properties of a potent and selective, small molecule inhibitor of PI3K-γ, IPI-549, and provide data to support the therapeutic potential of breaking tumor immune tolerance through PI3K-γ inhibition. Results: Discovery efforts identified a highly selective inhibitor of PI3K-γ, IPI-549, with pharmaceutical properties suitable for further development. Binding studies with IPI-549 revealed a KD value of 0.29 nM for PI3K-γ with >58-fold weaker binding affinity for the other Class I PI3K isoforms. Enzymatic assays utilizing physiological ATP concentrations (3 mM) confirmed the selectivity of IPI-549 for PI3K-γ (>200-fold) over other Class I PI3K isoforms. Cellular assays designed to assess individual Class I PI3K isoform activity demonstrated that IPI-549 is highly potent and specific for PI3K-γ (IC50 of 1.2 nM; >140-fold selectivity). Further selectivity screening revealed that IPI-549 is selective for PI3K-γ over other protein and lipid kinases, receptors, ion channels, and transporters. In vitro functional assays demonstrated that IPI-549 blocked bone marrow derived M2 murine macrophage polarization in response to IL-4 and MCSF1, but did not inhibit ConA-induced T-cell activation. These data indicate the potential for IPI-549 to block immune suppressive macrophage development but not T-cell activity. Pharmacokinetic studies in mice demonstrated IPI-549 to be orally bioavailable with a long plasma half-life enabling selective inhibition of the PI3K-γ isoform relative to the other Class I PI3K isoforms. In an in vivo PI3K-γ-dependent neutrophil migration murine model, IPI-549 blocked neutrophil migration in a dose dependent manner. To evaluate the effect of PI3K-γ inhibition on tumor growth in an immunocompetent animal, IPI-549 was tested in murine syngeneic solid tumor models. Mice treated with IPI-549 demonstrated significant tumor growth inhibition in multiple syngeneic models. Studies to elucidate the mechanism of tumor growth inhibition indicated that IPI-549 affects immune suppressive myeloid cell numbers and/or function, leading to an increase in cytotoxic T-cell activity. Studies in nude or CD8 T-cell depleted mice demonstrated the T-cell dependence of IPI-549 mediated tumor growth inhibition. Finally, in vivo studies with IPI-549 in combination with immune checkpoint inhibitors showed increased tumor growth inhibition compared to either monotherapy. Conclusions: IPI-549 is a potent and selective inhibitor of PI3K-γ with pharmaceutical properties that allow for the selective inhibition of PI3K-γ in vivo. Our findings provide evidence that targeted inhibition of PI3K-γ by IPI-549 can restore antitumor immune responses and inhibit solid tumor growth in preclinical models. IND-enabling studies with IPI-549 are ongoing to support its initial clinical exploration in the setting of solid tumors. Citation Format: Jeffery Kutok, Janid Ali, Erin Brophy, Alfredo Castro, Jonathan DiNitto, Catherine Evans, Kerrie Faia, Stanley Goldstein, Nicole Kosmider, Andre Lescarbeau, Tao Liu, Christian Martin, Karen McGovern, Somarajan Nair, Melissa Pink, Jennifer Proctor, Matthew Rausch, Sujata Sharma, John Soglia, Jeremy Tchaicha, Martin Tremblay, Vivian Villegas, Katherine Walsh, Kerry White, David Winkler, Vito Palombella. The potent and selective phosphoinositide-3-kinase-gamma inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B029.

Abstract 554: Checkpoint blockade therapy is improved by altering the immune suppressive microenvironment with IPI-549, a potent and selective inhibitor of PI3K-gamma, in preclinical models

The phosphoinositide-3-kinase (PI3K) lipid kinases transduce signals in response to various stimuli in different cell types. PI3K-γ is predominantly expressed in leukocytes and not expressed in most epithelial tumors or sarcomas. Genetic studies highlight an important role for PI3K-γ in myeloid-derived cells that constitute a key component of the immune suppressive tumor microenvironment (Schmid et al. Canc Cell 2011). Targeting PI3K-γ could therefore alter the immune tumor microenvironment, enabling the immune system to attack tumor cells more effectively. We are developing IPI-549, an investigational small molecule inhibitor of PI3K-γ, and provide data to support the therapeutic potential of breaking tumor immune tolerance through PI3K-γ inhibition. IPI-549 is a potent and selective inhibitor of PI3K-γ with favorable pharmacological properties. In vitro functional assays demonstrated that IPI-549 blocked bone marrow derived M2 murine macrophage polarization, but did not affect M1 polarization. Oral administration of IPI-549 to tumor-bearing mice resulted in significant tumor growth inhibition in multiple syngeneic solid tumor models at PI3K-γ selective doses. Analysis of the tumor-associated immune cells demonstrated that IPI-549 treatment results in decreased immune suppressive myeloid cells and increased CD8+ T cells, suggesting enhanced anti-tumor immunity. To address the requirement for targeting myeloid cells by IPI-549, CD11b+ cells were depleted from a transplanted whole tumor Lewis Lung Carcinoma model and the effect of IPI-549 on limiting tumor growth was abrogated. In addition, a myeloid-infiltrated B16-GMCSF model, but not the isogenic B16 model without GMCSF, was responsive to IPI-549. Studies in immune-deficient mice or CD8 T-cell depleted tumor bearing mice demonstrated the T-cell dependence of IPI-549-mediated tumor growth inhibition. IPI-549 treatment also led to a significant reduction in lung metastases in the 4T1 and B16-GMCSF models. Importantly, in vivo studies with IPI-549 in combination with the immune checkpoint inhibitors anti-PD-1, anti-PDL-1 and anti-CTLA-4 showed increased tumor growth inhibition in multiple models compared to monotherapies alone. These data can inform combinations for future clinical trials. Our studies support a role for PI3K-γ in immune suppressive myeloid cells in the tumor microenvironment and provide evidence that targeted inhibition of PI3K-γ by IPI-549 can restore antitumor immune responses and inhibit tumor growth in preclinical models. A Phase 1 study evaluating IPI-549 as an orally administered therapeutic, as a single agent and in combination with an anti-PD-1 antibody therapy, in patients with selected solid tumors is expected to begin in early 2016. Citation Format: Olivier De Henau, Taha Merghoub, David Winkler, Sujata Sharma, Melissa Pink, Jeremy Tchaicha, Matthew Rausch, Jennifer Proctor, Nicole Kosmider, John Soglia, Vito Palombella, Jeffery L. Kutok, Jedd D. Wolchok, Karen McGovern. Checkpoint blockade therapy is improved by altering the immune suppressive microenvironment with IPI-549, a potent and selective inhibitor of PI3K-gamma, in preclinical models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 554.