Dual Targeting of Oncogenic Signaling and Immune Suppression in High-Risk T-ALL - ABSTRACT T-cell acute lymphoblastic leukemia (T-ALL) accounts for 10–15% of pediatric and up to 25% of adult ALL cases. Despite advances in leukemia treatment, adult T-ALL patients continue to face poor outcomes, with overall survival rates below 50%. Notably, Early T-cell Precursor (ETP)-ALL represents a particularly aggressive subtype with an especially poor prognosis. There has been little improvement in mortality or expansion of effective therapeutic options for T-ALL, even amidst recent progress in precision medicine and immunotherapy. Therefore, identifying novel 'druggable' molecular markers and developing new targeted therapies and combined therapies are immediate pressing issues in T-ALL. To address this unmet need, we have identified a novel signaling pathway involving ATAD2, regulated by the CK2/IKAROS axis, which plays a critical role in T-ALL cell cycle progression. Our preliminary data show that dual targeting of ATAD2 signaling and the CK2/IKAROS axis results in synergistic anti-leukemic effects in both in vitro and in vivo models. Furthermore, single-cell RNA sequencing (scRNA-seq) analysis of T-ALL patient samples revealed significant alterations in the tumor microenvironment (TME). Specifically, we observed selective expression of the immunomodulatory protein CD70 in malignant TALL cells, along with enrichment of tumor-associated macrophages (TAMs) and exhausted T cells at diagnosis compared to remission. Importantly, both the CK2/IKAROS axis and ATAD2 were found to directly regulate CD70 transcription. Based on these findings, we hypothesize that 1) inhibition of ATAD2 or CD70 can restore antitumor immunity and suppress leukemic progression by reprogramming the immunosuppressive TME in T-ALL; 2) targeting the CK2/IKAROS axis may enhance the efficacy of ATAD2 inhibitors and CD70-targeted therapies. To test the hypothesis, we will examine the anti-leukemic effects and TME remodeling induced by targeting ATAD2 or CD70 in vitro in high-risk T-ALL patient-derived samples, and in vivo in the DN3 murine T-ALL and the CEM human T-ALL xenograft models (Aim 1); we will also define the transcriptional regulation of ATAD2 and CD70 by IKAROS, and assess how CK2 inhibition via CX-4945 modulates their expression and reshapes the TME. We will also evaluate the potential of CK2/IKAROS axis inhibition to enhance the efficacy of ATAD2 inhibitors and CD70-directed immunotherapies in vitro and in vivo, including in patient-derived xenograft (PDX) models of highrisk T-ALL and ETP-ALL (Aim2). This study will provide mechanistic insight into the regulation and function of ATAD2 and CD70 in high-risk T-ALL and clarify the role of CK2/IKAROS signaling in their transcriptional control. We expect that single-agent targeting of ATAD2 or CD70 will yield measurable antileukemic activity, while combination therapy with CK2 inhibition will offer superior therapeutic benefit by concurrently suppressing gene expression and protein function. Ultimately, our findings may pave the way for a novel, mechanism-based combination therapy for high-risk T-ALL, particularly in patients harboring IKZF1 mutations, providing a strong foundation for future clinical translation. Also, because the complex, systemic immune cells and tumor cells interactions in the TME of T-ALL cannot be fully replicated in vitro or by computational models, the use of murine and PDX models is essential. Using these models allows us to evaluate cancer biology, underlying mechanisms, and systemic efficacy and safety before progressing to human clinical trials ultimately.
