Toll-like Receptor 1 Is a Candidate Therapeutic Target in Acute Myeloid Leukemia
Authors: Eriksson Mia , Peña Pablo , Chapellier Marion , Högberg Carl , Fioretos Thoas , Ebert Benjamin L , Järås Marcus 
Published: October 2019

Acute myeloid leukemia (AML) is a fatal disease that contains rare immature cells with self-renewal and leukemia-initiating capacity, known as leukemia stem cells (LSCs). Because current therapies are inefficient in eradicating LSCs, new therapies are warranted that efficiently target this cell population. One strategy being explored towards new therapies is identification of novel therapeutic targets on the cell surface of AML stem cells.

To identify a cell surface protein upregulated on LSC, we used flow cytometry to measure the expression level of 13 cell surface proteins on immature leukemic cells from 18 AML patients and bone marrow cells from 7 healthy controls. We identified Toll-like receptor 1 (TLR1) as significantly upregulated in the immature CD34+CD38-compartment compared to corresponding normal cells that were almost devoid of TLR1 expression. These findings are consistent with elevated TLR1 mRNA levels observed in MDS patients (Wei et al, Leukemia, 2013).

To evaluate the role of Tlr1 on immature leukemic cells, we performed shRNA-mediated inhibition of Tlr1 in MLL-AF9-expressing murine c-Kit+ leukemic cells. By using lentiviral vectors expressing the Tlr1-shRNAs along with a puromycin resistance gene, we identified two unique shRNAs that successfully suppressed the Tlr1 transcript and protein expression in comparison to a shRNA control. We next co-expressed the two Tlr1-shRNAs along with GFP in leukemia cells and monitored the percentage of GFP positive cells over time. Expression of the Tlr1-shRNAs resulted in strong depletion of the leukemic cells both in vitro and in vivo relative to the control shRNA. These findings suggest that TLR1 is important for the growth and survival of leukemic cells.

To further address the role of Tlr1 on leukemic cells, we stimulated the leukemic cells with Pam3CSK4, a specific Tlr1/2 agonist. Pam3CSK4 alone was added to in vitro cultures of leukemic cells for three days, leading to increased survival and a slight increased number of leukemic cells. However, flow cytometric analysis revealed a differentiation shift of cells stimulated with Pam3CSK4 indicated by a decreased expression of the immature cell surface marker c-Kit and an increased expression of the myeloid linage marker Mac-1. To evaluate how Pam3CSK4 affects LSCs, we added Pam3CSK4 to ex vivo-cultures of leukemic cells for 3 days and then transplanted the cells into sublethally irradiated mice. Blood samples after two weeks showed a decreased leukemic burden in mice receiving Pam3CSK4-stimulated cells compared to controls. These findings suggest that enforced TLR1/TLR2-signaling causes differentiation of LSCs.

In summary, this study demonstrates that TLR1 is upregulated on AML-stem cell enriched patient cells and that TLR1 expression is finely balanced to maintain LSCs. More specifically, our data suggest that the leukemic cells require Tlr1-expression for survival, but enhanced Tlr1/Tlr2-activation force the LSCs into differentiation. Hence, our study suggests that approaches aiming either for inhibition or enforced activation of TLR1 in AML should be explored further towards a potential new AML therapy. Collectively, we here identify TLR1 as a novel and promising candidate therapeutic target in AML.

Disclosures

No relevant conflicts of interest to declare.