CK2 Inhibitor CX4945 Shows Strong In Vivo Synergistic Anti-Leukemia Activity with Rapamycin in B-ALL Due to Enhanced Ikaros Mediated Repression of mTOR Pathway
Authors: Gowda Chandrika , Song Chunhua , Ding Yali , Dhanyamraju Pavan kumar , Iyer Soumya , Payne Jonathon , Bamme Yevgeniya , Sharma Arati , Payne Kimberly J. , Dovat Sinisa 
Published: November 2019

Casein Kinase II (CK2) is a pro-oncogenic protein that is overexpressed in various cancers including leukemia. Ikaros encodes a DNA-binding zinc finger protein that functions as a tumor suppressor. Loss of Ikaros activity (genetic alteration and functional inactivation) in high-risk B-ALL is associated with resistance to conventional chemotherapy and a poor prognosis. Ikaros binds to the promoters and/or upstream regulatory sequences of its target genes and regulates their transcription via chromatin remodeling. CK2 phosphorylates Ikaros and impairs its tumor suppressor functions and interferes with Ikaros' ability to repress cell cycle progression and the PI3K pathway. Targeted inhibition of CK2 using specific inhibitor CX4945 restores Ikaros tumor suppressor function in high-risk B-ALL even in cases with single allele Ikaros deletion. CX4945 showed strong in vivo anti-leukemia effect in high risk B-ALL patient derived xenograft models (Song et al, Blood 2015). CX4945 is currently in clinical trial for solid tumors. mTOR is the key component of the PI3K/AKT/mTOR pathway - a major signaling pathway that stimulates cellular proliferation in B-ALL. Rapamycin is a specific inhibitor of mTOR that is currently in clinical trial for the treatment of relapsed/refractory leukemia. Here we report in vivo efficacy of combination therapy of rapamycin with CX4945 using high risk B-ALL pre-clinical models and describe mechanism of synergistic action of CX4945 and rapamycin.
Methods and results: The therapeutic effect of the CX-4945, in combination with rapamycin was tested using three different patient-derived xenografts (PDX) from patients with high-risk B-ALL and its efficacy will be compared to single-drug treatment. All these leukemia samples were obtained from high risk pediatric B-ALL patients with high CK2 expression and one had IKZF1 haplo - deficiency. After engraftment following tail vein inoculation of leukemia cells into immunocompromised (NRG) mice, mice were treated with vehicle, rapamycin or CX4945 alone or combination of both CX4945 and rapamycin. Anti-tumor effect was measured by quantifying leukemia burden in bone marrow, spleen and peripheral blood by cell count and immune-phenotyping using flow cytometry. Overall survival was measured using Kaplan -Myers analysis in another cohort after similar treatment regimen.
The analysis of Ikaros' global genome-wide occupancy in the human B-ALL cell line, Nalm6, and in primary cells from patient with B-ALL, showed that Ikaros binds to the upstream regulatory element (URE) of MTOR gene. Further, Ikaros binding to MTOR promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary B-ALL cells. The role of Ikaros in regulating mTOR transcription in Nalm-6 B-ALL cell line was tested using gain-of-function and loss-of-function experiments. Ikaros knock-down with shRNA results in increased transcription of mTOR whereas overexpression of Ikaros by transduction of Nalm-6 cells with Ikaros-containing retrovirus resulted in sharp reduction of mTOR expression. Since Ikaros function in B-ALL is negatively regulated by pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 can regulate expression of mTOR. Over-expression of CK2 via retroviral transduction resulted in increased transcription of the mTOR mRNA, as measured by qRT-PCR, as well as increased overall expression of mTOR protein, as measured by western blot. Increased expression of CK2 was associated with a loss of Ikaros binding to the MTOR gene promoter. CK2 inhibition was also associated with reduced histone H3K9ac and H3K4me3 marks at the mTOR promoter suggesting that CK2 and Ikaros regulate mTOR transcription via chromatin remodeling.
In summary, these results establish in vivo anti-leukemia effect of combination therapy using rapamycin and CX4945 in high risk B-ALL PDX models. CK2 inhibition with CX4945 represses the mTOR pathway by enhancing Ikaros-mediated transcriptional repression of mTOR and PI3K pathway genes. Results provide the rationale for evaluating clinical use of CK2 inhibitors in combination with mTOR inhibitors in high risk leukemia with dysregulation of mTOR/PI3K pathway. The results of our study establish a new paradigm for targeting the transcriptional network that regulates expression of the gene in hyperactive oncogenic signaling pathways such as mTOR.

Disclosures
Payne: Elf Zone, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.