Notch signaling promotes chemoresistance in FLT3-amplified acute myeloid leukemia

From the Bernstein Group, Clinical Research Division

Upregulation of FLT3 with internal tandem repeats (FLT3-ITD) occurs in approximately one quarter of patients with de novo acute myeloid leukemia (AML). Patients with FLT3-ITD often have more aggressive disease and relatively poor prognosis. One reason for this is that these patients have higher rates of relapse after conventional therapies, including those targeting the increased FLT3 expression. The tumor microenvironment (TME) has been implicated in the resistance to chemotherapies. In particular, endothelial cells (EC) in the TME have been shown to enhance survival, dormancy, and pro-metastatic qualities in many cancers. Additionally, ECs can be used to support long term growth of hematopoietic stem cells and leukemia cells in vitro, implicating them as critical cells for cell survival. Quy Le of the Bernstein Group (Clinical Research Division) wanted to better understand whether ECs were responsible for increased chemo-resistance of FLT3-ITD AML cells. The results of his investigation were recently published in Leukemia.

The authors wanted to take advantage of the EC coculture technique that supports the growth of AML cells in vitro to determine if EC can help AML cells survive chemotherapy. They used AC220, a clinically relevant FLT3 inhibitor, to treat cells derived from three patients with FLT3-ITD AML in standard liquid culture, or in coculture with EC. After three days, the authors reaffirmed that EC gave AML samples a survival advantage, with two out of three samples surviving better in EC coculture than liquid culture. However, when these cultures were treated with AC220, all three samples survived much better when cultured with EC, suggesting that EC promote increased cancer cell survival. The authors extended these conditions for two weeks with seven patient-derived FLT3-ITD samples. Again, EC coculture promoted better survival in all samples after treatment with AC220 than standard liquid culture. To determine what pathways might be involved in chemoresistance, the authors used RNA-sequencing to gain an unbiased view of global gene signatures after two days of AC220 treatment compared to a vehicle control while in EC coculture. Using gene set enrichment analysis, which surveys gene expression changes to determine what signaling pathways they fall into, the authors found that genes associated with cell cycle negatively correlated with AC220 treatment but genes associated with cell surface interactions and cell-cell communication were positively correlated with treatment. These data suggest that the cancer cells were becoming quiescent but allowing for increased protection and retention of the cell niche in the TME, two things that allow cancer cells to remain dormant in the body until relapse. 

Notch signaling in the tumor microenvironment promotes AML precursor cell survival during chemotherapy. Blockade of Notch signaling reduces AML cell survival.
Notch signaling in the tumor microenvironment promotes AML precursor cell survival during chemotherapy. Blockade of Notch signaling reduces AML cell survival. Figure provided by Dr. Quy Le

Notch signaling is an important cell signaling pathway with many roles involved in cell-cell communication. Intriguingly, Notch signaling has been shown to play an important role in hematopoietic stem cell quiescence and retention within their microenvironment. The authors wondered whether Notch signaling could be playing a role in EC-mediated chemoresistance of AML cells. Using their RNA-seq data, they found that Notch-receptors were expressed regardless of treatment, but other pathway members were more highly expressed in cells treated with AC220 than control. To further understand the role of Notch signaling in chemoresistance, the authors treated 13 patient-derived FLT3-ITD samples with Notch receptor blocking antibodies or isotype controls, as well as AC220, in EC coculture. When analyzing all patient-derived samples as a group, there was a trend towards lower survival when treated with a Notch blocking antibody. However, FLT3-ITD samples can be stratified by their allelic ratio (AR), a measurement of both the abundance of malignant cells in the sample, as well as whether each cell has 0, 1, or 2 mutant alleles. A higher AR indicates a higher presence of FLT3-ITD in the sample. When samples were stratified by their AR (above 0.78 being “high”), blockade of Notch signaling within high AR FLT3-ITD samples resulted in a significant reduction in cell survival. As high FLT3-AR samples are associated with presence of FLT3/ITD in the AML precursor cells, the authors sorted CD34+ CD33- (precursor cells) and CD34+ CD33+ (AML cells) from a high AR patient-derived AML sample, and used these in their coculture method. AML precursors were significantly sensitive to Notch blockade during AC220 treatment, while their more differentiated CD33+ AML cell counterparts were not, suggesting that Notch signaling acts on the AML precursor cells to promote their survival and chemoresistance. Dr. Le, first author of the study explained the significance of their findings: "Frequent relapse in [FLT3-IDT] patients suggests that there exists a rare subset of leukemic cells, called leukemic stem cells (LSC), which are able to resist chemotherapy and are the source of recurrent disease. Understanding how LSCs survive therapeutic agents is critical to developing effective treatment for AML patients. Our work indicates a role of the endothelial cell niche in LSC drug resistance and suggest that Notch blockade, a clinically feasible approach, may provide a strategy to overcome this resistance."

This study was supported by the Hartwell Foundation.

UW/Fred Hutch Cancer Consortium members Brandon Hadland, Soheil Meshinchi, and Irwin Bernstein contributed to this work.

Le, Q, Hadland, B, Meshinchi S and Bernstein, I. 2020. Notch blockade overcomes endothelial cell-mediated resistance of FLT3/ITD-positive AML progenitors to AC220 treatment. Leukemia. doi: 10.1038/s41375-020-0893-y.