PI3K-δ and PI3K-γ Inhibition by IPI-145 Abrogates Immune Responses and Suppresses Activity in Autoimmune and Inflammatory Disease Models

SummaryPhosphoinositide-3 kinase (PI3K)-δ and PI3K-γ are preferentially expressed in immune cells, and inhibitors targeting these isoforms are hypothesized to have anti-inflammatory activity by affecting the adaptive and innate immune response. We report on a potent oral PI3K-δ and PI3K-γ inhibitor (IPI-145) and characterize this compound in biochemical, cellular, and in vivo assays. These studies demonstrate that IPI-145 exerts profound effects on adaptive and innate immunity by inhibiting B and T cell proliferation, blocking neutrophil migration, and inhibiting basophil activation. We explored the therapeutic value of combined PI3K-δ and PI3K-γ blockade, and IPI-145 showed potent activity in collagen-induced arthritis, ovalbumin-induced asthma, and systemic lupus erythematosus rodent models. These findings support the hypothesis that inhibition of immune function can be achieved through PI3K-δ and PI3K-γ blockade, potentially leading to significant therapeutic effects in multiple inflammatory, autoimmune, and hematologic diseases

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

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

<p><b>A Combination treatment of duvelisib plus dexamethasone or ibrutinib leads to sustained inhibition of pAKT</b>. Western blot analysis of DOHH2 (top) and SU-DHL-4 (bottom) treated with duvelisib (1 μM), dexamethasone (500 nM), ibrutinib (100nM) or the combination of duvelisib plus dexamethasone or ibrutinib for 6 hours or 24 hours and stained with pAKT (S473). <b>B. mTOR inhibition can suppress re-phosphorylation of pAKT.</b> SU-DHL-4 cells treated with duvelisib (1 μM), the pan-PI3K inhibitor GDC-0941(500 nM) or the ERK inhibitor SCH-772984 (500 nM) resulted in re-phosphorylation of pAKT (S473) at 24 hours, while combination with the PI3K/mTOR inhibitor PF-04691502 (500 nM) prevented re-phosphorylation of pAKT at 24 hours. <b>C. Re-phosphorylation of pAKT after duvelisib treatment is dependent on mTORC2</b>. DOHH2 (top) or SU-DHL-4 (bottom) treated with duvelisib (1 μM), mTOR1/2 inhibitor AZD-8055 (200 nM) or the mTORC1 inhibitor rapamycin (100 nM) for 1 hour (left) or following a 24 hour treatment with duvelisib (1 μM)(right). <b>D. Duvelisib combinations with dexamethasone and ibrutinib reduce activation of downstream effectors.</b> DOHH2 (top) and SU-DHL-4 (bottom) were treated with duvelisib, dexamethasone and ibrutinib as in Fig 3A. Western blot staining for downstream effectors pPRAS40 (T246), pP70S6K (T389) and pS6 (S235/236). <b>E. Combination of duvelisib with dexamethasone or ibrutinib leads to inhibition of cap-dependent translation</b>. DOHH2 (left) and SU-DHL-4 (right) treated as in Fig 3A and western blot stained for p4EBP1 (S65), c-MYC, peIF4E (s209) and cleaved PARP. The total eIF4E blots demonstrate that equal amounts of protein were loaded per lane.</p

Targeted deletion of CX3CR1 reveals a role for fractalkine in cardiac allograft rejection

Fractalkine (Fk) is a structurally unusual member of the chemokine family. To determine its role in vivo, we generated mice with a targeted disruption of CX(3)CR1, the receptor for Fk. CX(3)CR1(–/–) mice were phenotypically indistinguishable from wild-type mice in a pathogen-free environment. In response to antibody-induced glomerulonephritis, CX(3)CR1(–/–) and CX(3)CR1(+/+) mice had similar levels of proteinuria and injury. CX(3)CR1(–/–) and CX(3)CR1(+/+) mice also developed similar levels of disease in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. We performed heterotopic MHC class I/II cardiac transplants from BALB/c mice into C57BL/6 mice. In the absence of cyclosporin A (CsA), there was no difference in graft survival time between CX(3)CR1(–/–) and CX(3)CR1(+/+) recipient mice. However, in the presence of subtherapeutic levels of CsA, graft survival time was significantly increased in the CX(3)CR1(–/–) mice. Characterization of cells infiltrating the grafts revealed a selective reduction in natural killer cells in the CX(3)CR1(–/–) recipients in the absence of CsA and a reduction in macrophages, natural killer cells, and other leukocytes in the presence of CsA. We conclude that Fk plays an important role in graft rejection. The development of CX(3)CR1 antagonists may allow reductions in the doses of immunosuppressive drugs used in transplantation

IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway

The transcription factors interferon regulatory factor 3 (IRF3) and NF-kappaB are required for the expression of many genes involved in the innate immune response. Viral infection, or the binding of double-stranded RNA to Toll-like receptor 3, results in the coordinate activation of IRF3 and NF-kappaB. Activation of IRF3 requires signal-dependent phosphorylation, but little is known about the signaling pathway or kinases involved. Here we report that the noncanonical IkappaB kinase homologs, IkappaB kinase-epsilon (IKKepsilon) and TANK-binding kinase-1 (TBK1), which were previously implicated in NF-kappaB activation, are also essential components of the IRF3 signaling pathway. Thus, IKKepsilon and TBK1 have a pivotal role in coordinating the activation of IRF3 and NF-kappaB in the innate immune response

The phosphoinositide-3 kinase (PI3K)-δ,γ inhibitor, duvelisib shows preclinical synergy with multiple targeted therapies in hematologic malignancies - Fig 1

<p><b>A. Growth inhibition (GI₅₀) of duvelisib across the panel of twenty cell lines</b>. The median GI₅₀ across the cell line panel is 0.59 μM. In six cell lines (<b>A & B</b>) with the asterisks (RL, KARPAS-299, RPMI-8226, GRANTA-519, OCI-Ly7, OPM-2), the GI₅₀ failed to reach growth Inhibition levels of greater than fifty percent. <b>B. Growth inhibition (GI₅₀) of duvelisib across the panel of twenty cell lines grouped by tumor subtype</b>. The median GI₅₀ across the cell line panel is 0.59 μM. Cell lines are grouped according to tumor subtypes: Diffuse large B cell lymphoma (DLBCL) activated B cell (ABC) and germinal center B cell (GCB), follicular lymphoma (FL), T cell lymphoma (T cell), mantle cell lymphoma (MCL) and multiple myeloma (MM).</p

Activity of duvelisib in a panel of malignant hematologic cell lines.

<p>Activity of duvelisib in a panel of malignant hematologic cell lines.</p