ALBERT

Description: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy resulting from the transformation of T-cell progenitors. Although the prognosis of this disease has substantially improved due to the introduction of intensified chemotherapy, the clinical outcome of T-ALL patients with primary resistant or relapsed disease remains poor, indicating that further therapeutic improvement is urgently needed. We have previously demonstrated that activation of tyrosine kinase 2 (TYK2) contributes to aberrant survival of human T-ALL cells. TYK2 is a member of the Janus-activated kinase (JAK) tyrosine kinase family and our report was the first to implicate TYK2 in T-ALL pathogenesis. Indeed, our gene knockdown experiments showed TYK2 dependency in 14 of 16 (88%) T-ALL cell lines and 5 of 8 (63%) patient-derived T-ALL cells tested, suggesting that inhibition of TYK2 would be beneficial in most patients with T-ALL. Based on these findings, we investigated the therapeutic potential of a novel small-molecule TYK2 kinase inhibitor NDI-031301 in T-ALL. We found that NDI-031301 shows potent and selective inhibitory activity against TYK2 in a cellular context, because this compound strongly inhibited the growth of TYK2-transfomed Ba/F3 cells when compared to the JAK inhibitors tofacitinib and baricitinib, whereas Ba/F3 cells transformed by other tyrosine kinases showed decreased sensitivity to NDI-031301. NDI-031301 induced robust growth inhibition in each of 4 human T-ALL cell lines representing different molecular subtypes of the disease (DU.528, KOPT-K1, HPB-ALL and SKW-3), with IC50 values of 0.8186 - 2.380 μM after 72 hours of exposure. NDI-031301 treatment of human T-ALL cell lines resulted in induction of apoptosis that was not observed with tofacitinib and baricitinib. To elucidate the mechanism of apoptosis induced by NDI-031301 in T-ALL cells, we next investigated cellular signaling pathways that are associated with cell survival and specifically affected by TYK2 inhibition with NDI-031301. Western blotting analysis demonstrated that treatment with 3 μM of NDI-031301 resulted in reduction of STAT1 Tyr-701 phosphorylation and BCL2 levels in KOPT-K1 cells, consistent with our previous finding that TYK2 phosphorylates STAT1 and upregulates BCL2 expression in most T-ALL cells. Surprisingly, the treatment also uniquely led to activation of three mitogen-activated protein kinases (MAPKs), resulting in phosphorylation of ERK, SAPK/JNK and p38 MAPK coincident with PARP cleavage, which was not observed with tofacitinib and baricitinib. NDI-031301-mediated activation of SAPK/JNK and p38 MAPK pathways are likely mediated through inhibition of TYK2, because increased phosphorylation levels of SAPK/JNK and p38 MAPK were observed in the cells transfected with TYK2-targeting shRNAs, while the levels of ERK1/2 phosphorylation were not upregulated. Further investigation revealed that activation of p38 MAPK occurred within 1 hour of NDI-031301 treatment and was responsible for NDI-031301-induced T-ALL cell death, as pharmacologic inhibition of p38 MAPK by SB203580 partially rescued apoptosis induced by TYK2 inhibitor, while inhibition of ERK or SAPK/JNK showed no rescue effects. Finally, we found that daily oral administration of NDI-031301 at 100mg/kg BID to immunodeficient mice engrafted with KOPT-K1 T-ALL cells was well tolerated, and led to decreased tumor burden and a significant survival benefit. After 29 days of treatment, the mice receiving NDI-031301 had marked reductions in infiltration of leukemia cells into spleen and bone marrow by comparison with controls. Thus, our findings clearly support TYK2 inhibition with NDI-031301 or a related compound as a potential therapeutic strategy for patients with T-ALL, and also raise the possibility that enhancing p38 MAPK activation in T-ALL cells may be an approach to accentuate its anti-leukemic activity. Disclosures Masse: Nimbus Therapeutics: Employment. Miao:Nimbus Therapeutics: Employment. Rocnik:Nimbus Therapeutics: Employment. Kapeller:Nimbus Therapeutics: Employment.