The transcription factor Nrf2 (nuclear factor (erythroid-derived 2)-like 2) and the kinase AMPK (AMP-activated protein kinase) participate in the cellular adaptive response to redox or energy stress. Despite accumulating evidence for positive cooperativity between both proteins, information about direct post-translational modification of Nrf2 by AMPK in living cells is scarce. Here, MS-based analysis of immunoprecipitated Nrf2 revealed serine 374, 408 and 433 in human Nrf2 to be hyperphosphorylated as a function of activated AMPK. A direct phosphate-transfer by AMPK to those sites was indicated by in vitro kinase assays with recombinant proteins as well as interaction of AMPK and Nrf2 in cells, evident by co-immunoprecipitation. Mutation of serine 374, 408 and 433 to alanine did not markedly affect half-life, nuclear accumulation or induction of reporter gene expression upon Nrf2 activation with sulforaphane. However, some selected endogenous Nrf2 target genes responded with decreased induction when the identified phosphosites were mutated, whereas others remained unaffected. Notably, the genes susceptible to the mutation of the phosphorylation sites in Nrf2 consistently showed reduced induction in AMPKα1 −/−cells. Overall, our data reveal AMPK-triggered phosphorylation of Nrf2 at three serine residues, apparently determining the extent of transactivation of selected target genes.

Leukemic cutaneous T‐cell lymphomas (L‐CTCL) are lymphoproliferative disorders of skin‐homing mature T‐cells causing severe symptoms and high mortality through chronic inflammation, tissue destruction, and serious infections. Despite numerous genomic sequencing efforts, recurrent driver mutations have not been identified, but chromosomal losses and gains are frequent and dominant. We integrated genomic landscape analyses with innovative pharmacologic interference studies to identify key vulnerable nodes in L‐CTCL. We detected copy number gains of loci containing the STAT3/5 oncogenes in 74% (n = 17/23) of L‐CTCL, which correlated with the increased clonal T‐cell count in the blood. Dual inhibition of STAT3/5 using small‐molecule degraders and multi‐kinase blockers abolished L‐CTCL cell growth in vitro and ex vivo, whereby PAK kinase inhibition was specifically selective for L‐CTCL patient cells carrying STAT3/5 gains. Importantly, the PAK inhibitor FRAx597 demonstrated encouraging anti‐leukemic activity in vivo by inhibiting tumor growth and disease dissemination in intradermally xenografted mice. We conclude that STAT3/5 and PAK kinase interaction represents a new therapeutic node to be further explored in L‐CTCL.

Constitutive activation of FLT3 by ITD mutations is one of the most common genetic aberrations in AML, present in ~1/3 of cases. Patients harboring FLT3-ITD display worse clinical outcomes. The integration and advancement of FLT3 TKI in AML treatment provided significant therapeutic improvement. However, due to the emergence of resistance mechanisms, FLT3-ITD+ AML remains a clinical challenge. We performed an unbiased drug screen to identify 18 compounds as particularly efficacious against FLT3-ITD+ AML. Among these, we characterized two investigational compounds, WS6 and ispinesib, and two approved drugs, ponatinib and cabozantinib, in depth. We found that WS6, although not yet investigated in oncology, shows a similar mechanism and potency as ponatinib and cabozantinib. Interestingly, ispinesib and cabozantinib prevent activation of AXL, a key driver and mechanism of drug resistance in FLT3-ITD+ AML patients. We further investigated synergies between the selected compounds and found that combination treatment with ispinesib and cabozantinib or ponatinib shows high synergy in FLT3-ITD+ AML cell lines and patient samples. Together, we suggest WS6, ispinesib, ponatinib and cabozantinib as novel options for targeting FLT3-ITD+ AML. Whether combinatorial tyrosine kinase and kinesin spindle blockade is effective in eradicating neoplastic (stem) cells in FLT3-ITD+ AML remains to be determined in clinical trials.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T-cell cancer. Hotspot mutations in JAK-STAT pathway members IL7R, JAK1 and JAK3 were analyzed in depth. However, the role of STAT5A or STAT5B mutations promoting their hyperactivation is poorly understood in the context of T-cell cancer initiation and acute leukemia progression. Importantly, the driver mutation STAT5BN642H encodes the most frequent activating STAT5 variant in T-ALL associated with poor prognosis. Here, we show that hyperactive STAT5 promotes early T-cell progenitor (ETP)-ALL-like cancer in mice and upregulated genes involved in T-cell receptor signaling (TCR), even in absence of surface TCR promoting. Importantly, these genes were also overexpressed in human T-ALL and other STAT5-dependent T-cell cancers. Moreover, human T-ALL cells were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers. Thus, we define STAT5 target genes in T-ALL that promote pre-TCR signaling mimicry. We propose therapeutic targeting using selective ZAP70 or STAT3/5 inhibitors in a subgroup of T-ALL patients with prominent IL-7R-JAK1/3-STAT5 activity.

NK/T-cell lymphoma (NKTCL) and γδ T-cell non-Hodgkin lymphomas (γδ T-NHL) are highly aggressive lymphomas that lack rationally designed therapies and rely on repurposed chemotherapeutics from other hematological cancers. Histone deacetylases (HDACs) have been targeted in a range of malignancies, including T-cell lymphomas. This study represents exploratory findings of HDAC6 inhibition in NKTCL and γδ T-NHL through a second-generation inhibitor NN-429. With nanomolar in vitro HDAC6 potency and high in vitro and in cellulo selectivity for HDAC6, NN-429 also exhibited long residence time and improved pharmacokinetic properties in contrast to older generation inhibitors. Following unique selective cytotoxicity towards γδ T-NHL and NKTCL, NN-429 demonstrated a synergistic relationship with the clinical agent etoposide and potential synergies with doxorubicin, cytarabine, and SNS-032 in these disease models, opening an avenue for combination treatment strategies.