IRAK4 Inhibitor Research Articles

S135: CRISPR/CAS9 EDITING REVEALS DEPENDENCE OF HUMAN RICHTER SYNDROME AND MURINE CLL CELLS ON SIGNALS FROM B CELL RECEPTOR, CXCR4 RECEPTOR AND MACROPHAGES BUT NOT FROM TOLL-LIKE RECEPTORS IN VIVO

Background: Numerous signals from the microenvironment have been identified that can increase the survival or induce the proliferation of chronic lymphocytic leukemia (CLL) cells in vitro. These signals typically represent various secreted or cell surface ligands that are expressed by different cell types present in the lymph node tumor microenvironment, such as T cells, macrophages and stromal cells, or molecules that would be expected to be released by apoptotic cells, such as apoptosis associated autoantigens or CpG-unmethylated mitochondrial DNA. However, the extent to which these different signals contribute to the growth and survival of the leukemic cells in vivo has still not been fully established. Aims: To determine the relevance of signals from Toll-like receptors (TLRs), the B cell receptor (BCR) and the chemokine receptor CXCR4 for the growth of murine Eμ-TCL1 CLL cells and human Richter Syndrome (RS) patient-derived xenograft (PDX) cells in vivo. Methods: To understand the impact of pharmacological inhibition of the TLR pathway, immunocompetent C57BL/6 or immunodeficient NSG mice were transplanted with murine Eμ-TCL1 CLL or human RS-PDX cells, respectively, and were treated with an inhibitor of the kinase IRAK4 or vehicle control. To determine the effects of genetic disruption of the TLR, BCR and CXCR4 pathways, the IRAK4, IgM heavy chain (IGHM) and CXCR4 genes were disrupted by CRISPR/Cas9 editing in the murine Eμ-TCL1 CLL cells or the human RS-PDX lines RS9737, RS1316 and IP867/17 prior to transplantation. The effects of genetic disruption of these pathways on the growth of the malignant cells in vivo were investigated by analyzing the proportion of mutant and wild type alleles in different anatomical compartments of the transplanted mice. Results: Treatment with the IRAK4 inhibitor significantly prolonged the survival of C57BL/6 mice transplanted with murine Eμ-TCL1 CLL cells and significantly delayed the growth of the human RS-PDX lines RS9737 and RS1316 xenografted in immunodeficient NSG mice. However, genetic disruption of IRAK4 in the human RS-PDX lines RS9737, RS1316 and IP867/17 or of the TLR adaptor MyD88 in the murine Eμ-TCL1 CLL cells did not result in negative selection of these cells in vivo, suggesting that these tumors do not receive or do not rely on TLR signals for their growth and that the therapeutic activity of the IRAK4 inhibitor is not caused by disruption of TLR signaling in the malignant cells themselves. In contrast, genetic disruption of the IGHM or CXCR4 gene was associated with significantly reduced growth of these cells compared to their wild type counterparts and resulted in almost complete disappearance of the mutated cells at later timepoints following transplantation. Analysis of the effects of the IRAK4 inhibitor on other cell types from the tumor microenvironment revealed a significant reduction in the number of macrophages, which in co-culture experiments were shown to strongly protect human Richter syndrome and murine Eμ-TCL1 leukemia cells from spontaneous apoptosis. Summary/Conclusion: These data provide evidence that signals from the BCR, CXCR4 and macrophages support the growth and survival of CLL and RS cells in vivo and argue against a role for TLR signals in the pathogenesis of CLL. In addition, they suggest that targeting the TLR pathway may potentially provide a therapeutic benefit in CLL, but that this benefit would be derived from inhibition of TLR signaling in monocytes and macrophages rather than inhibition of TLR signaling in the malignant cells themselves.

P1298: THE IRAK-4 INHIBITOR EMAVUSERTIB (CA-4948) FOR THE TREATMENT OF PRIMARY CNS LYMPHOMA

Background: Primary central nervous system lymphoma (PCNSL) is an aggressive brain tumor accounting for 3% of all CNS malignancies and is usually associated with poor prognosis. 5-year survival is less than 30% with current treatment options. Standard of care treatment includes induction with high-dose methotrexate based chemoimmunotherapy followed by consolidation with autologous stem cell transplant or whole brain radiation. However, more than half of PCNSL patients are not eligible for this type of intensive therapeutic intervention. Novel treatment strategies with better efficacy and toxicity profile are urgently needed. Toll-like receptor signaling pathway via MyD88/IRAK-4 signalosome is constitutively active in PCNSL secondary to MYD88 L265P mutation and represents an excellent therapeutic target. Emavusertib (CA-4948) is an oral first-in-class small molecule inhibitor of IRAK4 that has demonstrated clinical activity in patients with systemic Non-Hodgkin’s Lymphoma, however it has not been evaluated in the CNS space. Aims: In this study we examine translational and clinical therapeutic applicability and efficacy of emavusertib for the treatment of PCNSL. Methods: Using multiparameter immunohistochemistry (IHC) and proteomics analysis, IRAK-1, IRAK-4, and NF-κB pathway activation were examined in patient PCNSL tissues as compared to normal controls. Plasma, cerebrospinal fluid, and brain tissue were assessed via UPLC-MS/MS for emavusertib drug concentration following single oral high dose in a murine model. Using an aggressive preclinical A20 immunocompetent CNSL model, preclinical therapeutic activity of emavusertib was evaluated with assessment of downstream biomarker response to treatment including phospho-NF-κB, phospho-ERK1/2, and phospho-P38 in control and treated animals by multi-parameter IHC. We also report therapeutic response in a relapsed/refractory PCNSL patient enrolled in a Phase I/II clinical trial (TakeAim) assessing the combination of emavusertib and Ibrutinib. Results: Our data confirm elevated IRAK-1, IRAK-4, and NF-κB signaling in patient PCNSL. We also demonstrate that emavusertib can achieve therapeutically relevant concentrations in the brain parenchyma and shows single-agent activity in an aggressive preclinical model of PCNSL. We further confirm decreased ERK1/2, MAPK and NF-κB activation in response to preclinical emavusertib treatment, indicative of downstream effect of IRAK-4 inhibition. Finally, one patient who could not tolerate standard therapies and was resistant to Ibrutinib achieved complete response when treated with the combination of emavusertib and Ibrutinib, providing early clinical evidence of CNS penetration and anti-tumor activity of emavusertib. Summary/Conclusion: Herein we confirm for the first time that targeted inhibition of IRAK-4 with the oral small molecule inhibitor emavusertib demonstrates preclinical and clinical therapeutic activity against CNS lymphoma. Further evaluation of this agent for the treatment of PCNSL is warranted.

Respiratory protective effects of Korean Red Ginseng in a mouse model of particulate matter 4-induced airway inflammation

BackgroundAir pollution has led to an increased exposure of all living organisms to fine dust. Therefore, research efforts are being made to devise preventive and therapeutic remedies against fine dust-induced chronic diseases. MethodsResearch of the respiratory protective effects of KRG extract in a particulate matter (PM; aerodynamic diameter of <4 μm) plus diesel exhaust particle (DEP) (PM4+D)-induced airway inflammation model. Nitric oxide production, expression of pro-inflammatory mediators and cytokines, and IRAK-1, TAK-1, and MAPK pathways were examined in PM4-stimulated MH-S cells. BALB/c mice exposed to PM4+D mixture by intranasal tracheal injection three times a day for 12 days at 3 day intervals and KRGE were administered orally for 12 days. Histological of lung and trachea, and immune cell subtype analyses were performed. Expression of pro-inflammatory mediators and cytokines in bronchoalveolar lavage fluid (BALF) and lung were measured. Immunohistofluorescence staining for IRAK-1 localization in lung were also evaluated. ResultsKRGE inhibited the production of nitric oxide, the expression of pro-inflammatory mediators and cytokines, and expression and phosphorylation of all downstream factors of NF-κB, including IRAK-1 and MAPK/AP1 pathway in PM4-stimulated MH-S cells. KRGE suppressed inflammatory cell infiltration and number of immune cells, histopathologic damage, and inflammatory symptoms in the BALF and lungs induced by PM4+D; these included increased alveolar wall thickness, accumulation of collagen fibers, and TNF-α, MIP2, CXCL-1, IL-1α, and IL-17 cytokine release. Moreover, PM4 participates induce alveolar macrophage death and interleukin-1α release by associating with IRAK-1 localization was also potently inhibited by KRGE in the lungs of PM4+D-induced airway inflammation model. KRGE suppresses airway inflammatory responses, including granulocyte infiltration into the airway, by regulating the expression of chemokines and inflammatory cytokines via inhibition of IRAK-1 and MAPK pathway. Conclusion: Our results indicate the potential of KRGE to serve as an effective therapeutic agent against airway inflammation and respiratory diseases.

Phase I trial of CA-4948, an IRAK4 inhibitor, in combination with FOLFOX/PD-1 inhibitor +/- trastuzumab for untreated unresectable gastric and esophageal cancer.

TPS4168 Background: Activated NFκB has been linked to aggressive phenotype, poor survival outcomes and resistance to chemotherapy in multiple gastrointestinal cancers including gastroesophageal cancer (GEC). Preclinical studies established that: 1) Genotoxic stress incurred by chemotherapy induces TLR9, which signals through IRAK4 to drive pro-survival NFκB signaling; 2) The survival mechanism through IRAK4 is independent of cancer types and mutational profiles based on colorectal and pancreatic cancer models; and 3) IRAK4 inhibition reduces tumor desmoplasia and revitalizes intratumoral T cells, setting the stage for successful combination with immune checkpoint inhibitors in a highly aggressive autochthonous pancreatic cancer mouse model. These data combined provide a strong rationale to add CA-4948 to systemic therapy for multiple advanced gastrointestinal malignancies, where resistance to chemotherapy is inevitable and benefit of PD-1 inhibitors is limited to small population. CA-4948 is a novel, first-in-class reversible inhibitor of IRAK4. In a phase I trial, patients with relapsed/refractory hematologic malignancies tolerated CA-4948 monotherapy well with mild fatigue, neutropenia, and nausea as most common adverse events. Recommended phase 2 dose (RP2D) was determined as 300 mg orally twice daily. CA-4948 has not been tested in combination with cytotoxic chemotherapy or immune checkpoint inhibitors for solid tumors in clinic. We hypothesize that inhibition of IRAK4 with CA-4948 will potentiate the effect of immune checkpoint inhibitor while deepening the efficacy of cytotoxic chemotherapy in GEC. Methods: This is a phase I trial of CA-4948 in combination with FOLFOX/PD-1 inhibitor with or without trastuzumab for unresectable GEC. During Dose Escalation, we will investigate CA-4948 in combination with FOLFOX/nivolumab by BOIN algorithm evaluating 4 different dose levels. Starting dose of CA-4948 for Part A will be 200 mg twice daily. Once RP2D is determined, the study will proceed to Dose Expansion, including Cohorts A and B. Cohort A will enroll up to 12 patients with HER2 negative disease at the RP2D of CA-4948 determined at the Dose Escalation phase. Cohort B will investigate CA-4948 in combination with FOLFOX/pembrolizumab and trastuzumab. The initial 6 patients in Cohort B will be considered safety lead-in to confirm the safety and tolerability at the RP2D, followed by additional patients, up to 12 patients treated at the RP2D. The primary objective is to determine the safety and RP2D of CA-4948 in combination with FOLFOX/PD-1 inhibitor with or without trastuzumab. Secondary objectives are to determine the preliminary efficacy of the combination. Correlative studies to evaluate pharmacodynamic effects and to identify biomarkers associated with disease response are planned. Clinical trial information: NCT05187182.

CA-4948 for the treatment of melanoma brain metastasis.

2011 Background: Melanoma is a growing clinical health concern where the incidence has doubled over the last 30 years. A major driver of melanoma associated deaths is the development of brain metastases (MBM). It is estimated that 40-60% of patients with metastatic melanoma will develop MBM. Current treatment strategies have a great local control rate, but do not prevent recurrent disease, with a distant intracranial failure rate of 50% at 1 year. Novel treatments and an improved understanding of the MBM tumor microenvironment are needed. Toll-like receptor signaling is activated in cutaneous melanoma, triggering innate inflammatory activation downstream through the adapter proteins MyD88 and interleukin (IL)-1 receptor-associated kinase (IRAK-1 and -4). Increased expression of IRAK-4 stimulates transcription of multiple cellular kinases and transcription factors, and production of inflammatory chemokines and cytokines. This places IRAK-4 as a central driver of both intrinsic tumorigenicity and immune evasion in melanoma. In this study we examine IRAK-4 activation in MBM, as well as preclinical responsiveness to CA-4948, an oral first-in-class small molecule inhibitor of IRAK-4 that has demonstrated clinical activity and has an acceptable safety in cancer patients. Methods: Using multiparameter IHC and proteomics analysis IRAK-1, IRAK-4, and NF-κB pathway activation were examined in patient MBM tissues and compared to normal controls. Plasma, cerebrospinal fluid, and brain tissue were assessed via UPLC-MS/MS for CA-4948 drug concentration following single oral high dose in a murine model. Using an aggressive preclinical model of MBM, B16F10, downstream biomarker response to treatment including phospho-NF-κB, phospho-ERK1/2, and phospho-P38 was measured in control and CA-4948 treated animals using multi-parameter IHC. Survival response was also tested using the B16F10 model. Results: Our data confirm elevated IRAK-1, IRAK-4, and NF-κB signaling in patient MBM. We also demonstrate that CA-4948 is capable of achieving therapeutically relevant concentrations in the brain parenchyma, and shows single agent activity in an aggressive preclinical model of MBM. We further confirm decreased ERK1/2, MAPK and NF-κB activation in response to CA-4948 treatment. Conclusions: We present here for the first time that IRAK-4 is a strong candidate for targeted therapy in MBM. We further validate CNS penetrance of the oral IRAK-4 inhibitor CA-4948, where single agent markedly reduces the proliferative capacity of aggressive preclinical MBM, resulting in improved survival. These data warrant further investigation of CA-4948 for the treatment of melanoma brain metastases, with far reaching potential as an adjunct to standard of care.

A rock in the toll road: IRAK4 inhibition of TLR4-MyD88 signaling as potential treatment for addiction

Birth is preceded by inflammation at the fetal/maternal interface. Additionally, the newborn experiences stimuli that under any other circumstance could elicit an immune response. It is unknown, however, whether birth elicits an inflammatory response in the newborn that extends to the brain. Moreover, it is unknown whether birth mode may alter such a response. To study these questions, we first measured corticosterone and pro- and anti-inflammatory cytokines in plasma of mouse offspring at several timepoints spaced closely before and after a vaginal or Cesarean birth. We found highest levels of IL-6 one day before birth and surges in corticosterone and IL-10 just after birth, regardless of birth mode. We next examined the neuroimmune response by measuring cytokine mRNA expression and microglial number and morphology in the paraventricular nucleus of the hypothalamus and hippocampus around the time of birth. We found a marked increase in TNF-α expression in both brain regions a day after birth, and rapid increases in microglial cell number in the first three days postnatal, with subtle differences by birth mode. To test whether the association between birth and cytokine production or expansion of microglia is causal, we manipulated birth timing. Remarkably, advancing birth by a day advanced the increases in all of the markers tested. Thus, birth triggers an immune response in the body and brain of offspring. Our results may provide a mechanism for effects of birth (e.g., acute changes in cell death and neural activation) previously reported in the newborn brain.

A Pan-Cancer Analysis of IRAK1 Expression and Their Association With Immunotherapy Response.

IRAK1 is an active kinase which plays a critical role in IL-1/TLR signaling pathway involved in inflammation and innate immune response. Recently, increasing evidence supports a potential role of IRAK1 in cancer progression. However, no immunological pan-cancer analysis of IRAK1 is available. We aimed to explore the prognostic value and the immunological functions of IRAK1. A series of datasets including The Cancer Genome Atlas, GEPIA2, cBioPortal, HPA, TIMER2.0 were performed to explore the oncogenic and immunological roles of IRAK1, including the relationship between IRAK1 and prognosis, genetic mutation, GO and KEGG enrichment pathway analysis, immune state of different tumors, The results showed that IRAK1 levels were upregulated in more than 20 types of cancers compared to the normal tissues. IRAK1 expression was associated with poorer prognosis in different cancer types. For the most frequent DNA alteration of IRAK1 is amplification. And the result of the enrichment analysis suggested that IRAK1 related to immune checkpoint pathway in cancer. IRAK1 inhibitor pacritinib inhibit proliferation and upregulate PD-L1 expression in different cancer cell lines. Moreover, the patients who receiving anti-PD-L1 therapy with low IRAK1 expression had a better prognosis, and the objective response rate to anti-PD-L1 therapy was higher in the low IRAK1 group than in the high IRAK1 group in IMvigor210 cohort. Our study reveals that IRAK1 can function as a prognostic marker in various malignant tumors. And pacritinib upregulated PD-L1 expression in several cancer cell lines, which indicating that IRAK1 can be used as a reliable marker to predict the efficacy of immunotherapy.

Inhibition of IRAK4 dysregulates SARS-CoV-2 spike protein-induced macrophage inflammatory and glycolytic reprogramming.

Escalated innate immunity plays a critical role in SARS-CoV-2 pathology; however, the molecular mechanism is incompletely understood. Thus, we aim to characterize the molecular mechanism by which SARS-CoV-2 Spike protein advances human macrophage (Mϴ) inflammatory and glycolytic phenotypes and uncover novel therapeutic strategies. We found that human Mϴs exposed to Spike protein activate IRAK4 phosphorylation. Blockade of IRAK4 in Spike protein-stimulated Mϴs nullifies signaling of IRAK4, AKT, and baseline p38 without affecting ERK and NF-κB activation. Intriguingly, IRAK4 inhibitor (IRAK4i) rescues the SARS-CoV-2-induced cytotoxic effect in ACE2+HEK 293 cells. Moreover, the inflammatory reprogramming of Mϴs by Spike protein was blunted by IRAK4i through IRF5 and IRF7, along with the reduction of monokines, IL-6, IL-8, TNFα, and CCL2. Notably, in Spike protein-stimulated Mϴs, suppression of the inflammatory markers by IRAK4i was coupled with the rebalancing of oxidative phosphorylation over metabolic activity. This metabolic adaptation promoted by IRAK4i in Spike protein-activated Mϴs was shown to be in part through constraining PFKBF3, HIF1α, cMYC, LDHA, lactate expression, and reversal of citrate and succinate buildup. IRAK4 knockdown could comparably impair Spike protein-enhanced inflammatory and metabolic imprints in human Mϴs as those treated with ACE2, TLR2, and TLR7 siRNA. Extending these results, in murine models, where human SARS-CoV-2 Spike protein was not recognized by mouse ACE2, TLRs were responsible for the inflammatory and glycolytic responses instigated by Spike protein and were dysregulated by IRAK4i therapy. In conclusion, IRAK4i may be a promising strategy for severe COVID-19 patients by counter-regulating ACE2 and TLR-mediated Mϴ hyperactivation.Graphical abstract IRAK4i therapy counteracts Mϴ inflammatory and glycolytic reprogramming triggered by Spike protein. This study illustrates that SARS-CoV-2 Spike protein activates IRAK4 signaling via ACE2 as well as TLR2 and TLR7 sensing in human Mϴs. Remarkably, IRAK4i treatment can dysregulate both ACE-dependent and independent (via TLR sensing) SARS-CoV-2 Spike protein-activated inflammatory and metabolic imprints.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-022-04329-8.

Anticancer activity of IRAK-4 inhibitors against canine lymphoid malignancies.

The interleukin-1 receptor-related kinase 4 (IRAK4), downstream of myd88, plays an essential role in hyperactive TLR signalling seen in some B-cell lymphomas. In particular, efficient IRAK4 inhibitors of activated B-cell subtype of human diffuse large B-Cell lymphoma (DLBCL) are being developed. However, the anticancer effect of IRAK-4 inhibitors in veterinary medicine has not been elucidated. It is therefore explored in this study involving the GL-1 and CL-1 canine lymphoma cell lines in vitro. MyD88 expression was analysed using polymerase chain reaction. GL-1 and CL-1 cells were subjected to concentration- and time-dependent treatment with an IRAK-4 inhibitor and assessed for viability, TLR signalling association and apoptosis using a cell counting Kit-8 assay, Western blotting and flow cytometry. The GL-1 and CL-1 cells exhibited enhanced MyD88 expression, however, canine peripheral blood mononuclear cells (cPBMCs) did not. The IRAK-4 inhibitor reduced cell viability in a dose- and time-dependent manner, significantly reduced the phosphorylation of molecules associated with TLR signalling at IC50 such as IRAK1, IRAK4, NF-κB and STAT3, and induced apoptosis in GL-1 and CL-1 cells. The anticancer effect of the IRAK-4 inhibitor on canine lymphoma cells is mediated by apoptosis via downregulation of TLR signalling.

Pharmacological inhibition of IRAK1 and IRAK4 prevents endothelial inflammation and atherosclerosis in ApoE-/- mice

Inflammation associated endothelial dysfunction represents a pivotal contributor to atherosclerosis. Increasingly, evidence has demonstrated that interleukin 1 receptor (IL1-R) / toll-like receptor (TLR) signaling participates in the development of atherosclerosis. Recent large-scale clinical trials have supported the therapeutic potential of anti-inflammatory therapies targeting IL-1β and IL-6 in reducing atherosclerosis. The present study examined the pharmacological effects of IL-1R-associated kinase 1 and 4 inhibitors (IRAK1/4i) in regulating inflammation of the endothelium and atherosclerosis. We demonstrate that dual pharmacological inhibition of IRAK1 and IRAK4 by an IRAK1/4i is more effective against LPS induced endothelial inflammation, compared with IRAK1 inhibitor or IRAK4 inhibitor monotherapy. IRAK1/4i showed little endothelial cell toxicity at concentrations from 1 μM up to 10 μM. Inhibition of IRAK1/4 reduced endothelial activation induced by LPS in vitro as evidenced by attenuated monocyte adhesion to the endothelium. Mechanistically, blockade of IRAK1/4 ameliorated the transcriptional activity of NF-κB. To assess the pharmacological effects of IRAK1/4i on atherosclerosis in vivo, ApoE-/- mice were orally administered IRAK1/4i (20 mg/kg/d) for 8 weeks. We show that IRAK1/4i reduced atherosclerotic lesion size in the aortic sinus and increased hepatic LDLR protein levels as well as lowered LDL-C level, without affecting other lipid parameters or glucose tolerance. Taken together, our findings demonstrate that dual pharmacological inhibition of IRAK1 and IRAK4 attenuates endothelial inflammation, lowers LDL-C levels and reduces atherosclerosis. Our study reinforces the evolving standing of anti-inflammatory approaches in cardiovascular therapeutics.

Abstract P073: Pharmacological inhibition of IRAK4 with CA-4948 is beneficial in marginal zone lymphoma models with secondary resistance to PI3K and BTK inhibitors

Abstract Background. Marginal zone lymphoma (MZL) is a heterogeneous B-cell malignancy for which no standard treatment exists. B-cell receptor (BCR) signaling dysregulation is a hallmark of MZL and can mimic antigen dependent BCR activation. However, single treatment with BCR signaling inhibitors shows limited complete response rate and relapsing patients. IRAK4 is a protein kinase downstream the Toll-like receptor signaling. CA-4948 is the first-in-class inhibitor of IRAK4 and it is in phase 1/2 studies for patients with relapsed/refractory clinical studies with favorable activity in lymphomas and myeloid neoplasms. Here, we assessed IRAK4 inhibition with CA-4948 against a panel of MZL cell lines in which we have developed secondary resistance to inhibitors of PI3K (idelalisib, copanlisib) or BTK (ibrutinib). Methods. Models with secondary resistance were developed by continuous exposing cell lines derived from splenic MZL (Karpas 1718 and VL51) to high doses of drugs for long period of times. Resistant and parental lines were exposed to increasing doses of CA-4948, alone or in combination with compounds they are resistant to. Cell viability was tested by MTT assay (72h treatment). Synergy of combinations was evaluated according to the Chou-Talalay combination index (CI), as well as potency and efficacy, estimated according to the MuSyC algorithm. Results. The parental Karpas1718, bearing a MYD88 L265P mutation, had an IC50 of 3.29 µM for CA-4948 as single agent, while the parental VL51 and all resistant models presented IC50 values in the range of 19-35 µM. IRAK4 inhibition with CA-4948 had strong benefit when combined to downstream BCR inhibitors. In resistant cells, CA-4948 was strongly synergistic with idelalisib, ibrutinib and, at lesser extent, with copanlisib. CA-4948 in combination with ibrutinib was strongly synergistic especially in the VL51 ibrutinib resistant model compared to the parental one. CA-4948 (from 1 to 5 µM) recovered sensitivity to ibrutinib at IC50 values close to the parental condition. Similarly, CA-4948 in combination with idelalisib was also synergistic and increased sensitivity to idelalisib in idelalisib resistant VL51. Less benefit was seen adding CA-4948 to copanlisib: the combination was synergistic in VL51 parental cell, but with only weak efficacy. In the Karpas1718, CA-4948 was synergistic either in idelalisib resistant or in parental, with improved benefit in the latter, in which synergistic effect was observed when combined with both idelalisib and ibrutinib. The synergistic activity of CA-4948 was mainly due to improved efficacy rather than potency of the combinatorial partners. Conclusions. Our results in MZL cell lines show that the IRAK4 inhibitor CA-4948 is synergistic with small molecules targeting BCR signaling, especially with idelalisib and ibrutinib. Data also suggest that concentrations of CA-4948 comparable to those achieved in patients might overcome or reduce secondary resistance to the tested tyrosine kinase inhibitors. Citation Format: Francesca Guidetti, Alberto J. Arribas, Filippo Spriano, Laura Barnabei, Reinhard von Roemeling, Elizabeth Martinez, Emanuele Zucca, Francesco Bertoni. Pharmacological inhibition of IRAK4 with CA-4948 is beneficial in marginal zone lymphoma models with secondary resistance to PI3K and BTK inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P073.

Abstract P243: The IRAK4 inhibitor CA-4948 demonstrates antitumor activity in a preclinical model of CNS lymphoma

Abstract Background: Central nervous system (CNS) lymphoma, both primary and secondary disease, carries the worst prognosis of all Non-Hodgkin’s lymphoma with a 5 year survival of less than 30% with current therapeutic options. Recent studies have shown constitutive activation of both NF-κB and MAPK signaling pathways in the maintenance and progression of CNS lymphoma. This often coincides with mutations in MYD88. IRAK4 is a serine/threonine kinase that mediates interleukin 1 receptor (IL-1R) and Toll-like receptor (TLR) signaling downstream of MYD88, and is a novel therapeutic target for this disease. CA-4948 is an oral first-in-class small molecule inhibitor of IRAK4 that has demonstrated clinical activity in patients with systemic Non-Hodgkin’s Lymphoma. Our study goal was to evaluate CA-4948 efficacy in a syngeneic preclinical model of primary CNS B cell lymphoma (A20), supporting future clinical investigation of this agent in this difficult to treat disease. Materials and Methods: Plasma, cerebrospinal fluid (CSF), and brain tissue (both naïve and tumor bearing) were assessed for CA-4948 drug concentration following single high-dose oral administration using UPLC-MS/MS. Biomarker expression, including NF-κB and MAPK, was measured in control and CA-4948 treated brain tissue resected from tumor-bearing animals using multi-parameter immunohistochemistry and 3D modeling of cleared tissue. Dose-dependent survival responses were also measured using the A20 preclinical model of primary CNS lymphoma. Results: While brain concentrations of CA-4948 remained low with respect to plasma (~4-5%), therapeutic dose levels were attained in both CSF and brain tissue (&amp;gt;502 nM). No significant difference between naïve and A20 tumor bearing brain tissue was observed, indicating the blood brain barrier remained intact in this model. Biomarker analyses revealed significant downregulation of both MAPK and NF-κB signaling pathways in response to CA-4948, as evidenced by reduced protein levels of phospho-p38Thr180/Tyr182, phospho-ERK1/2Thr202/Tyr204, and phospho-p65Ser536 in both the tumor cells and the tumor microenvironment. Finally, CA-4948 treatment produced a dose-dependent survival response in syngeneic animals harboring CNS A20 tumors. Conclusions: CA-4948 is able to reach therapeutic dose levels in the CNS in a preclinical murine model of CNS lymphoma, producing significant and dose-dependent anti-tumor activity and survival advantage. These data support further evaluation of CA-4948 in clinical investigation and treatment of patients with CNS lymphoma. Citation Format: Christina A. von Roemeling, Bently P. Doonan, Lan Hoang-Minh, Han W. Tun, Elizabeth Martinez, Raul Soikes, Reinhard von Roemeling, Duane A. Mitchell. The IRAK4 inhibitor CA-4948 demonstrates antitumor activity in a preclinical model of CNS lymphoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P243.

Long-Term Treatment with Pacritinib on a Compassionate Use Basis in Patients with Advanced Myelofibrosis

Long-Term Treatment with Pacritinib on a Compassionate Use Basis in Patients with Advanced Myelofibrosis

Interleukin-1/Toll-like Receptor Inhibition Can Restore the Disrupted Bone Marrow Microenvironment in Mouse Model of Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by bone marrow (BM) failure and associated with aging. We have previously shown that reversal of BM microenvironment (BMME) dysfunction in MDS mitigates MDS associated marrow failure and delays progression to acute leukemia. However, the exact mechanisms driving BMME dysfunction in MDS remain unknown. We recently reported that interleukin-1 (IL1) Receptor Type1 (IL1R1) signaling is a driver in myeloid bias via disruption of BMME in aging. In addition, we have found that IL1R1 signaling is involved in disease progression of AML. Therefore, to assess the role of IL1R1 signaling in MDS associated BMME dysfunction and marrow failure, we employed an age appropriate murine transplant model for MDS utilizing NUP98-HOXD13 (NHD13) transgenic mice.Methods: BM cells (NHD13 transgenic or wild type (WT), 7 weeks) and competitor cells were transplanted into irradiated aged recipients (WT or IL1R1 KO, 60 weeks), and subsequently monitored for development of marrow failure. When marrow failure developed, mice were euthanized and peripheral blood, BM, BM extracellular fluid (BMEF), and collagenase-1 digested bone associated cells were analyzed including flow cytometry, colony forming units (CFU) assay, and cytokine analyses. Next, BM from NHD13 (8-10 weeks) and competitor cells were transplanted into lethally irradiated aged recipients (WT, 50-60 weeks). At onset of marrow failure, mice were treated with inhibitors of IL1/Toll-like receptor signaling (IL1R antagonist, MCC950, or IL1R-associated kinase 4 protein (IRAK4) inhibitor) for fourteen days, and then euthanized and analyzed as above. Finally, we evaluated cytokine profile in the BM serum from the patients with MDS and normal donors.Results: Transplant of NHD13 BM cells into aged IL1R1 wt recipients (NHD13→IL1R1 wt) was not associated with a significant difference in survival rates or levels of NHD13 engraftment compared to NHD13 into IL1R1 ko recipients (NHD13→IL1R1 ko). IL1R1 wt developed macrocytic anemia compared to IL1R1 ko recipient (Hb 11.3±0.57 v.s 13.1±0.42 g/dL, n=12 and 9, p<0.05). In CFU-C assays, NHD13→IL1R1 wt and NHD13→IL1R1 ko demonstrated similar levels of CFU-activity whereas CFU-fibroblast (CFU-F) assays of IL1R1 wt recipients demonstrated lower numbers of large colonies (reported to contain highly proliferative mesenchymal stem cells (MSC)). Flow cytometry analysis of hematopoietic stem and progenitor cells (HSPC) population in IL1R1 wt recipients showed increased myeloid progenitors and decreased long-term HSC (LT-HSC) compared to IL1R1 ko recipients. Cytokine/chemokine profiles revealed that the inflammatory cytokines (IL-6 and TNFα) and macrophage activating cytokines (MCP-1 and M-CSF) were significantly decreased in NHD13→IL1R1 ko compared to NHD13→IL1R1 wt. Next, we treated MDS model mice with inhibitors. The pharmacological targeting of IL1R1 signaling in vivo was associated with decreased NHD13 cell burden and improvement of macrocytic anemia. CFU-C assays demonstrated some decreases in NHD13 CFU capacity post-treatment. Interestingly, non-NHD13 HSPCs assessed by CFU capacity increased in IRAK4 inhibitor treated mice. Consistent with this, flow cytometric analyses of HSPC pools demonstrated decreased NHD13 HSPCs (LT-HSC and granulocyte-monocyte progenitor cells) and increased non-NHD13 HSCs and myeloid progenitors compared to vehicle group. BMME cell populations showed that arteriolar endothelial cells and MSCs were also affected by drug treatment and both IL1R antagonist and MCC950 increased the number of large CFU-F colonies. Analysis of BMEF revealed the decreased IFNγ and IL-18 and upregulated M-CSF in MCC950 treated group. The cytokines of human BM serum revealed higher concentrations of soluble formed IL1R1, IL-18, CXCL1, and osteopontin in MDS compared to young or aged normal donors.Conclusions: Collectively, our findings demonstrate that IL1R1 signaling alters the BMME and contributes to the disease phenotype of MDS and that the effects of targeting IL1R1 pharmacologically have differing effects based on the modality of inhibition as well as the cell population. IL1R1 signaling can be a promising target to alleviate the complexity of MDS via improving inflammatory status in BMME. DisclosuresNo relevant conflicts of interest to declare.

IRAK4 inhibitor mitigates joint inflammation by rebalancing metabolism malfunction in RA macrophages and fibroblasts

Recent studies show a connection between glycolysis and inflammatory response in rheumatoid arthritis (RA) macrophages (MΦs) and fibroblasts (FLS). Yet, it is unclear which pathways could be targeted to rebalance RA MΦs and FLS metabolic reprogramming. To identify novel targets that could normalize RA metabolic reprogramming, TLR7-mediated immunometabolism was characterized in RA MΦs, FLS and experimental arthritis. We uncovered that GLUT1, HIF1α, cMYC, LDHA and lactate were responsible for the TLR7-potentiated metabolic rewiring in RA MΦs and FLS, which was negated by IRAK4i. While in RA FLS, HK2 was uniquely expanded by TLR7 and negated by IRAK4i. Conversely, TLR7-driven hypermetabolism, non-oxidative PPP (CARKL) and oxidative phosphorylation (PPARγ) were narrowly dysregulated in TLR7-activated RA MΦs and FLS and was reversed by IRAK4i. Consistently, IRAK4i therapy disrupted arthritis mediated by miR-Let7b/TLR7 along with impairing a broad-range of glycolytic intermediates, GLUT1, HIF1α, cMYC, HK2, PFKFB3, PKM2, PDK1 and RAPTOR. Notably, inhibition of the mutually upregulated glycolytic metabolites, HIF1α and cMYC, was capable of mitigating TLR7-induced inflammatory imprint in RA MΦs and FLS. In keeping with IRAK4i, treatment with HIF1i and cMYCi intercepted TLR7-enhanced IRF5 and IRF7 in RA MΦs, distinct from RA FLS. Interestingly, in RA MΦs and FLS, IRAK4i counteracted TLR7-induced CARKL reduction in line with HIF1i. Whereas, cMYCi in concordance with IRAK4i, overturned oxidative phosphorylation via PPARγ in TLR7-activated RA MΦs and FLS. The blockade of IRAK4 and its interconnected intermediates can rebalance the metabolic malfunction by obstructing glycolytic and inflammatory phenotypes in RA MΦs and FLS.

MPN-053: Tumor-Intrinsic IRAKI-Dependent Inflammation Signaling Induces MDSC to Establish Immune Evasion in FGFRI-Driven Hematological Malignancies

Context: Stem cell leukemia/lymphoma syndrome (SCLL) presents as a myeloproliferative disease that can progress to acute myeloid leukemia and is associated with the coincident development of B-cell and T-cell lymphomas. SCLL is driven by the constitutive activation of FGFR1, as a result of chromosome translocations, with poor outcome. Objective: In this study, we use this model to investigate how FGFR1 drives IRAK1-dependent inflammatory signaling in the modulation of antitumor immunity. Design: CRISPR/Cas9 approaches were used to generate SCLL cells null for IRAK1, which were introduced into syngeneic hosts through tail vein injection. Development of the disease and changes in immune cell composition were monitored using flow cytometry. Results: In SCLL, FGFR1 activation silences miR-146b-5p through DNMT1-mediated promoter methylation, which derepresses the downstream target IRAK1. IRAK1 knockout (KO) in SCLL cells leads to the loss of typical rapid progression when xenografted into immunocompetent mice, and the mice remained disease-free. IRAK1 in this system has no effect on cell cycle progression or apoptosis, and robust growth of the KO cells in immunodeficient mice suggested an effect on immune surveillance. Depletion of T-cells in immunocompetent mice restored leukemogenesis of the KO cells. Analysis of the immune cell profile in mice transplanted with the IRAK1-null cells shows that there is a reduction in levels of myeloid-derived suppressor cells (MDSCs) with a concomitant increase in CD4+/CD8+ T-cell levels. Immunoprofiling of a panel of secreted cytokines and chemokines showed that activation of INF-γ is specifically impaired in the KO cells. In vitro and in vivo expression assays and engraftment into IFNGR1-null mice showed SCLL leukemia cells produced INF-γ, not only promoting intrinsic PD-L1 expression but also participating in the induction of MDSCs to establish immune evasion. Inhibition of IRAK1 using pacritinib suppresses leukemogenesis with impaired induction of MDSCs and attenuated suppression of CD4+/CD8+ T-cells. Conclusions: IRAK1 orchestrates a previously unknown FGFR1-directed immune escape mechanism in SCLL, through induction of IFN-γ, and targeting IRAK1 may provide a means of suppressing tumor growth in this syndrome by restoring immune surveillance. This work was supported by grant CA076167 from the National Institutes of Health.

USP13-mediated IRAK4 deubiquitination disrupts the pathological symptoms of lipopolysaccharides-induced sepsis

Ubiquitin-specific peptidase 13 (USP13) has been reported to participate in tumorigenesis, cell cycle arrest, endoplasmic reticulum-associated degradation, and immune responses. Here, we explored the function of USP13 in pro-inflammatory cytokine production of macrophages and its role in mouse sepsis model. Primary bone-marrow-derived macrophages (BMDMs) isolated from wild type (WT) and USP13MKO mice were treated by lipopolysaccharides (LPS), IL-4, toll-like receptors (TLRs) agonists, and IRAK4 inhibitor to profile the inflammatory responses with different genotypes. Mouse sepsis model (WT and USP13MKO) created by intraperitoneal injection with LPS plus d-galactosamine was used to assess septic shock-induced survival and lung inflammation. Flow cytometry, qRT-PCT, Western blot, and ELISA were performed to detect pro-inflammatory production and macrophage polarization. USP13 was a key regulator of IRAK4 deubiquitination in BMDMs and its myeloid specific deficiency contributed to LPS-induced pro-inflammatory response and septic symptoms. IRAK4 inhibitor co-administration improved in LPS-induced inflammatory responses in both BMDMs and septic mice. USP13 negatively regulates LPS-induced sepsis shock by targeting IRAK4. In summary, targeting USP13-IRAK4 axis might be a potential therapeutic strategy for the treatment of inflammation in sepsis shock.

Pharmacological inhibition of IRAK1 attenuates colitis‐induced tumorigenesis in mice by inhibiting the inflammatory response and epithelial–mesenchymal transition

Colorectal cancer (CRC) is the third most common type of cancer. Here, we studied the inhibitory effect of IRAK1 and IRAK4 as a preventive strategy using a colitis-induced tumorigenesis mouse model. CRC clinical data were obtained from the Gene Expression Omnibus (GEO). An experimental inflammation-dependent CRC model was induced by treatment with azoxymethane (AOM) and then dextran sodium sulfate (DSS) in C57BL/6 mice. Mice were administered an IRAK1/4 inhibitor by intraperitoneal injection at 3 mg/kg twice each week for 9 weeks. The IRAK1/4 inhibitor attenuated histological changes and prevented tumor growth. Tumor-associated proteins, including p65 and Ki-67, were downregulated by the IRAK1/4 inhibitor in AOM/DSS-treated mice. Additionally, IRAK1/4 inhibitor administration effectively decreased the expression of inflammatory cytokines. Furthermore, we observed that IRAK1/4 inhibitor treatment attenuated colitis-induced tumorigenesis by inhibiting epithelial-mesenchymal transition. These observations indicate that inhibition of IRAK1 and IRAK4 may suppress experimental colitis-induced tumorigenesis by inhibiting inflammatory responses and epithelial-mesenchymal transition.

Abstract 385: Identification of NF-kappaB phospho-p50 as a predictive biomarker for IRAK4 inhibitor CA-4948 in patients with non-Hodgkin's lymphoma

Abstract Background: IRAK4, a serine/threonine kinase, was identified as a therapeutic target in hematological malignancies. IRAK4-mediated activation of NF-kappaB could play an important role in NF-kappaB-regulated survival and chemoresistance of cancer cells. CA-4948, a first-in-class small molecule inhibitor of IRAK4, has been tested in Phase 1 clinical trial with evidence of clinical activity in Non-Hodgkin's Lymphoma (NHL) patients. To support the development of companion diagnostic for CA-4948, we developed an immunohistochemical (IHC) assay and explored expression of potential biomarkers of response to CA-4948 in tumor biopsy samples obtained from NHL patients. Materials and Methods: IHC assay was developed and used for analysis of NF-kappaB p-p50 and p-IRAK1 expression in tumor biopsy samples obtained from 14 patients with NHL. Patients were defined according to their clinical response to CA-4948 treatment: stable disease (SD), 6 cases and progressive disease (PD), 8 cases. Results: We found nuclear and/or cytoplasmic expression of NF-kappaB p-p50 in all 6 SD cases treated with 50 mg QD (2 cases, tumor regression), 50 mg BID (1 case), 200 mg BID (1 case, tumor regression) and 400 mg BID (2 cases). Expression of NF-kappaB p-p50 was not detected in 7 of 8 cases with PD including patients treated with 50 mg QD (1 case), 100 mg QD (1 case), 100 mg BID (3 cases), 200 mg BID (1 case) and 400 mg BID (1 case). We found statistically significant correlation between expression of NF-kappaB p-p50 in tumor biopsy and SD in NHL patients treated with CA-4948 (p&amp;lt;0.05). Analysis of NF-kappaB p-p50 expression in paired tumor biopsy samples (3 cases) collected before and after the treatment with CA-4948 revealed a significant downregulation of NF-kappaB p-p50 expression in tumors obtained from CA-4948-treated NHL patients. In support of our in vivo findings, our in vitro experiments demonstrated that expression of NF-kappaB p-p50 was depleted in 3D lymphoma organoids treated with a clinically relevant concentration of CA-4948. Our results support further development of NF-kappaB p-p50 as a potential predictive and pharmacodynamic biomarker of IRAK4 inhibitor CA-4948. We found that expression of p-IRAK1 in tumor biopsies was not correlated with clinical response. Conclusions: Although the cohort size is small, our findings suggest that expression of NF-kappaB p-p50 can serve as biomarker to predict SD in response to the treatment with IRAK4 inhibitor CA-4948 in NHL patients. NF-kappaB p-p50 selection strategy might be used in future clinical trials to identify NHL patients which are most likely to respond to CA-4948 in combination with chemotherapy or targeted therapeutics. Citation Format: Andrey Ugolkov, Maria Samson, Rosanna Hok, Reinhard von Roemeling, Robert Martell. Identification of NF-kappaB phospho-p50 as a predictive biomarker for IRAK4 inhibitor CA-4948 in patients with non-Hodgkin's lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 385.

Combination of IRAK4 Inhibitor CA-4948 with BCL2 Inhibitor Venetoclax Induces Tumor Regression in an ABC-DLBCL Xenograft Model

IRAK4 kinase activity is required for toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling in a variety of myeloid and lymphoid cell types. Recruitment of IRAK4 to these receptors and its subsequent activation is facilitated by the MYD88 adaptor protein, which is mutated in ~22% of diffuse large B-cell lymphoma (DLBCL) cases. The MYD88- L265P activating mutation is found in ~30% of the activated B-cell (ABC) and ~6% of germinal center B-cell (GCB) subtypes of DLBCL and leads to constitutive activation of NF-κB signaling that is associated with worse prognosis. In Waldenstrom macroglobulinemia (WM), the MYD88- L265P activating mutation is present in >90% of cases. Thus, the development of small molecule inhibitors targeting IRAK4 is an attractive anticancer strategy for MYD88 mutation-containing cancers such as DLBCL and WM.We are developing a novel IRAK4 inhibitor, CA-4948, as a therapeutic agent for non-Hodgkin lymphoma (NHL) and hematological cancers with dysregulated TLR/MYD88/IRAK4 signaling. CA-4948 is a reversible kinase inhibitor that modulates IRAK4 function in both the toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) signaling cascades, and demonstrates pharmacodynamic and antitumor activity in in vitro and in vivo nonclinical models. CA-4948 exhibits favorable DMPK properties, oral bioavailability, and is well tolerated in mice. Furthermore, CA-4948 was previously shown to exhibit dose-dependent efficacy in ABC-DLBCL MYD88 -L265P xenograft tumor models using cell lines OCI-LY3 and OCI-Ly10 and patient-derived tumors.Activating mutations in the B-cell receptor (BCR) signaling pathway are frequently present in various NHL subtypes, leading to activation of NF-kB signaling, and growth and survival pathways. Dysregulation of both TLR and BCR pathways suggest that targeting both pathways will be required for improved therapeutic responses in NHL. Here, we report efficacy results from combined treatment of CA-4948 and the BCL2 inhibitor venetoclax in an ABC-DLBCL xenograft tumor model.We performed an in vivo study in female SCID Beige mice implanted with the ABC-DLBCL cell line OCI-Ly10, which harbors TLR (MYD88 -L265P) and BCR (CD79A ITAM) pathway activating mutations. OCI-Ly10 tumor-bearing mice were treated with CA-4948 (50 mg/kg, qd, po), venetoclax (75 mg/kg, qd, po) or the combination for 21 continuous days. Single agent treatment of CA-4948 and venetoclax exhibited moderate tumor growth inhibition (TGI) of 63% and 71%, respectively, and the combination demonstrated tumor regression. The combination was tolerated at the end of treatment without body weight loss relative to predose weight. After the 21-day drug treatment period, the mice were given a 19-day dosing holiday. Within 5 days of the holiday rapid tumor growth occurred in the previously treated single drug-treated mice and this rapid growth continued for the remainder of the dosing holiday. In contrast, tumors from the combination drug-treated mice did not commence detectable regrowth until the 10th day of the drug holiday and proceeded with a slow growth rate for the subsequent 9 days.After the 19-day drug holiday, dosing of the respective drug treatments was reinstated to each cohort. After 7 days of re-dosing, the tumors in each cohort exhibited antitumor responses to respective drug treatments with the CA-4948 plus venetoclax combination again driving the tumors into regression. In summary, CA-4948 plus venetoclax was a well-tolerated drug combination that exhibited a much greater in vivo antitumor response as compared to the single agent treatments. These results underscore the therapeutic potential of targeted IRAK4 kinase inhibition by CA-4948 in combination with other targeted agents for the treatment of NHL. DisclosuresBooher:Curis, Inc: Employment, Equity Ownership. Dellarocca:Curis, Inc: Employment, Equity Ownership. Atoyan:Curis, Inc: Employment, Equity Ownership. Borek:Curis, Inc: Employment, Equity Ownership. Samson:Curis, Inc: Employment, Equity Ownership. Tuck:Curis, Inc: Employment, Equity Ownership.