Hoang-Minh, Lan

Abstract Purpose: An ongoing challenge in cancer is the management of primary and metastatic brain malignancies. This is partly due to restrictions of the blood-brain barrier and their unique microenvironment. These challenges are most evident in cancers such as lymphoma and melanoma, which are typically responsive to treatment in systemic locations but resistant when established in the brain. We propose interleukin-1 receptor-associated kinase-4 (IRAK-4) as a potential target across these diseases and describe the activity and mechanism of oral IRAK-4 inhibitor CA-4948. Experimental Design: Human primary central nervous system lymphoma (PCNSL) and melanoma brain metastases (MBM) samples were analyzed for expression of IRAK-4 and downstream transcription pathways. We next determined the central nervous system (CNS) applicability of CA-4948 in naïve and tumor-bearing mice using models of PCNSL and MBM. The mechanistic effect on tumors and the tumor microenvironment was then analyzed. Results: Human PCNSL and MBM have high expression of IRAK-4, IRAK-1, and nuclear factor kappa B (NF-κB). This increase in inflammation results in reflexive inhibitory signaling. Similar profiles are observed in immunocompetent murine models. Treatment of tumor-bearing animals with CA-4948 results in the downregulation of mitogen-activated protein kinase (MAPK) signaling in addition to decreased NF-κB. These intracellular changes are associated with a survival advantage. Conclusions: IRAK-4 is an attractive target in PCNSL and MBM. The inhibition of IRAK-4 with CA-4948 downregulates the expression of important transcription factors involved in tumor growth and proliferation. CA-4948 is currently being investigated in clinical trials for relapsed and refractory lymphoma and warrants further translation into PCNSL and MBM.

Abstract BACKGROUND PCNSL is an aggressive brain tumor accounting for 3% of all CNS malignancies and is associated with poor prognosis. 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 cannot tolerate this intensive therapeutic intervention. Novel treatments 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 IRAK-4 that demonstrates clinical activity in patients with systemic Non-Hodgkin’s Lymphoma, however it has not been evaluated in the CNS space. HYPOTHESIS: Emavusertib has anti-tumor activity against preclinical 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. Survival responses in preclinical PCNSL models were measured in response emavusertib treatment, along with in vivo tumor proliferation and downstream biomarker expression by multi-parameter IHC. RESULTS Our data confirm elevated IRAK-1, IRAK-4, and NF-κB signaling in patient PCNSL. We show that emavusertib can achieve therapeutically relevant concentrations in the brain and CSF. Emavusertib impairs tumor cell proliferation in vitro and in vivo, and shows dose-dependent single-agent activity in preclinical PCNSL models. We confirm decreased ERK1/2, MAPK and NF-κB activation, indicative of downstream IRAK-4 inhibition. CONCLUSIONS Our data supports that emavusertib demonstrates anti-tumor activity in the CNS space, warranting further preclinical and clinical evaluation of this agent for the treatment of PCNSL.

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 (>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.