Triggering Panoptosis in Acute Myeloid Leukemia Cells
Authors: Lewis Cameron , Runde Austin P , Zhang Jiwang , Breslin Peter 
Published: December 2024

Acute myeloid leukemia (AML), the most common hematologic cancer in humans, is characterized by the uncontrolled proliferation of leukemia blasts and insufficient hematopoiesis. With current frontline treatments, &gt;30% of patients still present with primary resistance (refractory) and nearly 40% of responding patients will develop secondary resistance (relapse); less than 30% of AML patients survive five years from diagnosis. Novel treatments are urgently needed to overcome drug resistance. Almost all conventional chemotherapies kill AML cells by inducing apoptosis, and most AML cells in relapsed/refractory (R/R) patients resist treatment by repressing apoptotic signaling. However, other programmed cell death (PCD) pathways are still intact. We speculate the apoptosis-resistant AML cells from R/R patients can be killed via induction of other PCD pathways included in a collective concept, PANoptosis.
AML cells arise from mutated hematopoietic stem/progenitor cells (HSPCs) and express innate immune-associated machinery. PANoptosis involves the parallel activation of pyroptosis, apoptosis, and necroptosis, and is triggered by innate immune signaling that induces assembly of the PANoptosome, a multiprotein complex. Within the PANoptosome, FADD-Caspase 8 mediates apoptosis, RIPK1-RIPK3 mediates necroptosis, and NLRP3-ASC-Caspase 1 mediates pyroptosis. The integrated and interconvertible activation of three types of PCD signaling in the PANoptosome makes it a unique target to kill AML cells. Caspase 8 is a modulator of the three types of PCD: activation induces apoptosis and/or pyroptosis, whereas inhibition promotes necroptosis. RIPK1, the master regulator of PANoptosis, is required for PANoptosome assembly and is stimulated by a diverse array of upstream effectors, including the Toll-like receptors (TLRs). In most cell types, two key mediators of innate immune signaling, TAK1 and TBK1, restrict PANoptosis. As Ser/Thr protein kinases, TAK1 and TBK1 phosphorylate RIPK1 thereby inhibiting PANoptosome assembly. Thus, pharmacologic blockade of TAK1 and/or TBK1 may sensitize AML cells to PANoptotic stimulation.
Our lab has previously reported that RIPK1 protein can barely be detected in healthy CD34+ HSPCs yet is highly expressed (RIPK1high) in AML cells from &gt;50% of primary patient samples and established human AML cell lines. PANoptotic signaling in RIPK1high AML cells is intact and thus such AML cells may be sensitive to PANoptotic stimulation. Pharmacologic blockade of TAK1 and/or TBK1 might be an effective way to kill drug-resistant AML cells and may be augmented by azacitidine (AZA), which could further stimulate PANoptosis by causing an accumulation of cytosolic dsDNA/dsRNA, activating endosomal TLRs. In an MLL-AF9-transformed murine AML model, we found that TAK1 is required for the survival of leukemia blasts, whereas TBK1 is essential for the survival of slow-cycling, Flt3+ leukemia stem cells.
To test whether induction of PANoptosis can kill AML cells, we first treated AML cell lines with TAK1- and/or TBK1-selective inhibitors with or without AZA. We found that TAK1 blockade and, to a lesser extent, TBK1 blockade induced PCD in RIPK1high AML cell lines, including MOLM-13 and MM6, but failed to do so in RIPK1low cell lines such as HL-60. The pro-apoptotic effects of TAK1 blockade were augmented by AZA in RIPK1high cell lines, where it was also determined that TAK1 blockade could rescue the cell-killing ability of half-dose cytarabine. TAK1 blockade also ablated the colony-forming ability of MOLM-13 cells. Interestingly, K562 cells (BCR-ABL1+) are RIPK1high yet resistant to TAK1/TBK1 blockade. Such information provides us with a rationale to investigate PANoptotic signaling in AML cells, to understand how this signaling is regulated and might be exploited therapeutically. Currently, we are escalating our TAK1/TBK1 blockade investigation to AML patient samples/xenograft models, to determine if this strategy is effective and can also be augmented by AZA. While TAK1 inhibitors have not been tested clinically, TBK1 inhibitors have been demonstrated to be safe/well-tolerated, and data from our Tbk1-knockout murine model suggest TBK1 blockade can be done without significantly impeding homeostatic hematopoiesis. We predict the results of our study will provide a solid conclusion to transfer our combination treatment regimen into a clinical trial for AML treatment.