Divergent Pro-Leukemic Effects of MYD88 and TICAM-1/TRIF Mediated Toll-Like Receptor Signaling

The goal of this study is to determine the role of Toll-like receptor (TLR) signaling in acute myeloid leukemia (AML). AML is a hematopoietic malignancy that predominately affects the elderly. The median age of AML diagnosis is 67 years old. The standard of care of AML is treatment with cytotoxic chemotherapy, which elderly patients are often unable to tolerate. To identify novel pathways as potential targets for treatment, a microarray on AML primary patient samples was performed to determine the expression of key inflammatory genes. This microarray, and other published datasets, showed that TLR are overexpressed in myelomonocytic (M4) and monocytic (M5) AML subtypes. TLRs are components of the innate immune system that sense and respond to inflammatory stimuli from infections or tissue injury. TLR signaling is mediated by one of two adaptor proteins, myeloid differentiation factor 88 (MYD88) or Toll/IL1 receptor (TIR) containing adaptor molecule 1 (TICAM-1). Inhibition of kinases downstream from these receptors inhibits MLL-AF9 leukemia cell growth. To investigate TLR signaling in the setting of AML, two models were employed. First, inducible genetic deletion of the Myd88 gene in MLL-AF9 leukemia cells was applied. Myd88 was determined to be required for leukemia cell stemness, colony forming potential, proliferation, resistance to chemotherapy, and leukemogenesis in vivo. Next TICAM-1 expression was suppressed by shRNA in human and murine models and found to have similar effects, protecting leukemia cell stemness, proliferation, colony forming potential, and leukemogenesis in vivo. Knockdown of downstream kinases, IL-1 receptor-associated kinase 1 for Myd88, and Tank-binding kinase for Ticam-1, failed to recapitulate these phenotypes. While the exact mechanism of Myd88 pro-leukemic function remains undetermined, TICAM-1 was shown to protect protein levels of receptor-interacting protein kinase 1 (RIPK1) and RIPK3, and TICAM-1 knockdown in human ML-2 or murine MLL-AF9 cells resulted in corresponding loss of RIPK1 and RIPK3. Together, these data indicate that TLR-associated adaptor proteins participate in pro-leukemia signaling pathways independently from TLR-associated kinases. These findings have implications for the treatment of AML and suggest that targeted disruption or inhibitions of MYD88 or TICAM-1-dependent signaling pathways might have clinical benefit

TAK1 and TBK1 are Differentially Required by GMP- and LMPP-like Leukemia Stem Cells

Acute myeloid leukemia (AML) encompasses a diverse group of cancers that originate in the blood-forming tissues of the bone marrow. Aside from the M3 subtype (PML-RARA+), 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 2-phase regimen of intensive chemotherapy (CTx) employing daunorubicin and cytarabine. 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. AML relapses when a pool of undetectable, CTx-resistant leukemia stem cells (LSCs) survives & proliferates after frontline CTx [1]. Notably, the poor performance status of many AML patients precludes use of the standard CTx regimen; while reduced-intensity CTx still offers therapeutic benefit, it is less effective at killing LSCs and, as a result, relapse is more likely. Goardon, et al. determined that AML patients harbor two types of LSCs: granulocyte-macrophage progenitor (GMP)-like LSCs and FLT3+ lymphoid-primed multipotential progenitor (LMPP)-like LSCs [2]. Eradication of both types of LSCs is necessary to maintain CR in AML. Our group and others have established that ~40% of AML patients express upregulated Toll-like receptor (TLR) signaling (TLR+). TLR+ disease is associated with specific genetic abnormalities, such as MLL rearrangements (MLL-r+), and is inversely associated with prognosis (Figure 1) [3,4]. TLR+ AML represents a challenging, treatment-sparse subset of an already difficult-to-treat disease. To study TLR+ AML, we utilize an MLL-r+ model using the MLL-AF9 oncogene. We have also demonstrated that both GMP- and LMPP-like LSCs require TLR-associated Ser/Thr protein kinases for their survival [5-7]. Specifically, GMP-like LSCs require TAK1 and LMPP-like LSCs require TBK1. The loss of either Tak1 or Tbk1 ablates the corresponding LSC pool and enriches for the opposite LSC pool in vitro and in vivo. Recently, our group determined that the genetic loss of Tak1 sensitizes mouse AML cells to TBK1 blockade in vitro. Strikingly, the loss of Tbk1 also seems to extend overall survival (OS) despite causing extramedullary AML. While mice given Tbk1NULL AML cells develop a subcutaneous tumor of AML cells (chloroma) near the pelvis, they survive longer than mice given control (Tbk1WT) AML cells. The clinical significance is unknown, but these data support our impression that the loss of Tbk1 forces AML cells to differentiate; this should be therapeutically favorable, as inducing the differentiation of AML cells is an effective treatment strategy. Theoretically, chloromas may form in Tbk1NULL AML due to the enrichment of GMP-like LSCs, which express higher levels of chemokine receptors. We hypothesize that the differentiation & eradication of LSCs can be induced by blocking TAK1/TBK1 in combination with standard CTx (and possibly targeted agents like Mylotarg®, Venclexta®, and/or Xospata®). We propose TAK1/TBK1 parallel blockade as augmentation to standard CTx, ideally allowing for a dose-reduction of CTx & promoting improved patient outcomes

Sensitizing Leukemia Stem Cells to NF-κB Inhibitor Treatment in Vivo by Inactivation of Both TNF and IL-1 Signaling

We previously reported that autocrine TNF-α (TNF) is responsible for JNK pathway activation in a subset of acute myeloid leukemia (AML) patient samples, providing a survival/proliferation signaling parallel to NF-κB in AML stem cells (LSCs). In this study, we report that most TNF-expressing AML cells (LCs) also express another pro-inflammatory cytokine, IL1β, which acts in a parallel manner. TNF was produced primarily by LSCs and leukemic progenitors (LPs), whereas IL1β was mainly produced by partially differentiated leukemic blasts (LBs). IL1β also stimulates an NF-κB-independent pro-survival and proliferation signal through activation of the JNK pathway. We determined that co-inhibition of signaling stimulated by both TNF and IL1β synergizes with NF-κB inhibition in eliminating LSCs both ex vivo and in vivo. Our studies show that such treatments are most effective in M4/5 subtypes of AML