Identification of the Atypical Protein Kinase WNK1 As a New Target in T-Cell Acute Lymphoblastic Leukemia

Landscape genome-wide profiling studies identified critical cellular pathways altered in T-cell Acute Lymphoblastic Leukemia (T-ALL). Recurrent genetic abnormalities occur in genes involved in lymphoid development, oncogenes or tumor suppressors, cytokine receptors, and chromatin remodeling. Rarely, in this disease, mutations affect targetable kinases while they can be activated without causative genetic events. Thus, integrating phosphoproteomics with genomic approaches may offer novel opportunities for target discovery and therapeutic interventions.

To do so, we developed a Luminex bead-based assay for the simultaneously profiling a 31 phosphorylated kinases panel to identify relevant kinases in T-ALL cell lines or primary samples. Among differentially phosphorylated proteins, WNK1 shared with CREB and ERK the highest difference in mean fluorescent intensity (DMFI) comparing T-ALL vs. T cells. Moreover, we observed that thymocytes lack WNK1 phosphorylation compared to leukemic cells suggesting a cancer-intrinsic activation mechanism for this pathway and a new role of this kinase in T-ALL.

WNKs (with no lysine = K) proteins (WNK1-4) are atypical serine-threonine kinases where the catalytic lysine, required for the ATP binding, is replaced by cysteine and located in the subdomain I instead of II. WNK proteins control ion fluxes transport across cellular membranes and stimulate the release/ recycling of carrier vesicles by the oxidative stress-responsive 1 (OSR1) and sterile20 related proline and alanine-rich kinase (SPAK) proteins. Considering that protein phosphorylation pathways involved in lymphoid differentiation are commonly correlated with gene transcription, we compared the expression pattern of WNK1-4 in T-ALL to other cancers by mining large databases. Our results indicated that WNK1 is highly expressed in T-ALL lymphoblasts, T-ALL cell lines, and patient-derived leukemia xenograft (PDLX). Patients with a high WNK1 expression are predominantly observed in patients with T-ALL that belong to the TAL/LMO subtype and positively correlated with the PTEN mutational status. Consistently, TAL1 rearranged cell lines, such as HSB2 and MOLT-16, were amongst the most sensitive to the pan-WNK-kinase inhibitor, WNK463, compared to non-TAL1 cases such as DND41 or PF382. As expected, the phosphorylation level of WNK1 (T60 and S382) and the downstream targets SPAK/OSR1 (S372/S325) decreased upon genetic or small molecules inhibition treatment in T-ALL cells.

In dependent cells, shRNA or sgRNA targeting WNK1 promoted polyploidy resulting in cell cycle arrest and T-ALL proliferation inhibition in vitro. Similarly, WNK463 treatment leads to dramatic morphometric changes such as incomplete cell division or chromosome segregation through mitotic spindles and abscission defects. This is consistent with the observation that in WNK1 deprived T-ALL cells, transcriptional changes quantified by RNA-Seq reflect the modulation of signatures associated with G2M checkpoint and mitotic spindle control. Furthermore, loss of WNK1 reduces the phosphorylation level of proteins required for the successful progression of mitosis such as p-ERK1/2 and p-CREB/p-ATF1 in WNK1-dependent T-ALL cell lines and primary samples. Collectively, these results suggest a role of WNK1 in controlling leukemia progression in TAL/LMO T-ALL.

Because of the potential translational relevance of our finding, we tested WNK463 efficacy in vivo in a T-ALL PDLX model. Mice exposed to WNK463 for 21 days at 1.5 mg/Kg/day showed a significant decrease in bone marrow infiltrating leukemia cells and a reduction in spleen sizes associated with a decrement in the phosphorylation level of WNK1 as assessed by pharmacodynamic studies. Not significant toxicities were seen in treated animals.

In conclusion, our work demonstrates that WNK1 kinase actively contributes to the development of leukemia. Moreover, due the lack of sequence similarity in the ATP binding domain with other kinases, the identification of a leukemia dependency on the atypical WNK1 kinases represents an exceptional opportunity for developing selective inhibitors for the T-ALL treatment.