TAK1 Blockade Plus Azacitidine Induces Apoptosis, Augments Cytarabine, and Ablates Clonogenicity in MLL-AF9+ Human AML Cell Lines

Acute myeloid leukemia (AML) encompasses a diverse group of cancers that originate in the blood-forming tissues of the bone marrow [1]. Aside from the PML-RARA+ subtype, AML carries a 5-year survival rate of 28% for patients 20+ years of age. AML is the most common cancer of the hematopoietic system and is slightly more common in biological males; the average age at diagnosis is 68 years. Standard frontline treatment for AML is a two-phase regimen of intensive chemotherapy (CTx) employing daunorubicin and cytarabine; etoposide may also be added in select regimens. Despite 60-70% of patients achieving complete remission (CR), at least half of CR-achieving patients experience relapse within 3 years from their diagnosis. Additionally, 30-40% of patients present with refractory AML, experiencing little to no benefit from frontline treatment. Nearly all CTx, including daunorubicin and cytarabine, rely on p53-mediated apoptosis to kill AML cells. However, especially in relapsed/refractory (R/R) cases, AML cells frequently harbor TP53 mutations and actively repress apoptotic machinery to resist chemotherapy [2]. However, other programmed cell death (PCD) pathways, such as pyroptosis and necroptosis, may remain intact. We speculate that apoptosis-resistant, R/R AML cells can be killed via induction of other PCD pathways included in a collective concept, PANoptosis [3]. AML cells arise from mutated hematopoietic stem/progenitor cells (HSPCs) and express innate immunity-associated machinery, including Toll-like receptors (TLRs); PANoptosis—the parallel activation of pyroptosis, apoptosis, and necroptosis—is triggered by innate immune signaling, which induces assembly of the PANoptosome. RIPK1, the master regulator of PANoptosis, is required for PANoptosome assembly. RIPK1 is stimulated by an array of upstream effectors including TLRs, which are upregulated in certain subsets of AML such as the notoriously hard-to-treat cases harboring the MLL-AF9 translocation [4]. Within the PANoptosome, NLRP3-ASC-Caspase 1 mediates pyroptosis, FADD-Caspase 8 mediates apoptosis, and RIPK1-RIPK3 mediates necroptosis. Two key transducers of innate immune signaling, the TAK1 and TBK1 Ser/Thr kinases, restrict PANoptosis by phosphorylating RIPK1, inhibiting PANoptosome assembly. Previous research from our lab shows that TAK1 and TBK1 are each differentially required by two distinct subtypes of AML stem cells [5,6]. Thus, we hypothesize that pharmacologic blockade of TAK1 and/or TBK1 (with HS-276 and GSK8612, respectively) could sensitize AML cells to PANoptotic stimulation—and do so selectively, as PANoptotic machinery is active in AML cells but not healthy HSPCs (Figure 1) [4]. Additionally, azacitidine (Vidaza®, Onureg®), a pyrimidine antimetabolite that promotes DNA hypomethylation by inhibiting DNMT1, could augment TAK1/TBK1 blockade as AZA may activate endosomal TLRs by causing an accumulation of cytosolic dsDNA/dsRNA [7].