The Role of Notch in Endothelial Cell-Mediated Protection of AML Precursors from Targeted Therapy

Background: AML is an aggressive hematologic malignancy that remains difficult to treat. A common mutation found in AML is FLT3-ITD, occurring in 15% of childhood AML. Although chemotherapy has successfully induced remission, patients with a high FLT3 ITD:WT allelic ratio (FLT3-AR) exhibit a high relapse rate, requiring hematopoietic stem cell transplantation to increase the chance of long-term remission. In this study, we demonstrate the requirement of ECs for survival of FLT3-ITD progenitors from primary pediatric AML specimens in the presence of AC220, a potent and selective inhibitor of FLT3. We further show that the Notch pathway plays a role in EC-mediated protection amongst patient samples with high FLT3-AR, suggesting the potential therapeutic use of Notch blockade in the treatment of this high-risk subset.

Results: To determine whether ECs confer protection to FLT3-ITD progenitors, we quantified the number of CFC present after 2 weeks of liquid culture or EC co-culture with AC220 (added at days 0, 3 and 7) from four AML specimens with high FLT3-AR (≥1). We used PCR to determine the presence of FLT3-ITD in individual CFC. We found that the numbers of FLT3-ITD CFC (p=0.007) and FLT3-WT CFC (p=0.044) were reduced in liquid culture compared to EC co-culture, suggesting that ECs mediate the survival of FLT3-ITD hematopoietic progenitors against the therapeutic treatment of AC220.

Previously, we demonstrated that ECs are critical for the growth and expansion of hematopoietic stem cells, which is dependent on the activation of Notch signaling. We asked whether Notch plays a role in EC-mediated protection of AML progenitors against AC220, using RNA-seq analysis on three FLT3-ITD-harboring AML. Among the significantly altered genes (FDR<0.05), we found an enrichment of Notch target genes that were expressed at significantly higher levels in AC220-treated cells compared to DMSO-treated cells, including HES1, HES4, NRARP, CDKN1A, CCND1, andGATA3, suggesting that Notch signaling may facilitate EC-mediated protection against AC220.

Next, we assessed the effect of inhibiting Notch signaling on AML progenitor survival during AC220 treatment in EC co-culture, using inhibitory antibodies specific to the Negative Regulatory Region (NRR) of both Notch1 and Notch2 receptors (anti-NRR1 and anti-NRR2; kindly provided by Chris Siebel, Genentech). We co-cultured bone marrow cells from eight patient specimens with low FLT3-AR (<1) and five patient specimens with high FLT3-AR (≥1), with ECs and briefly treated the co-cultures with Notch inhibitory antibodies or IgG1 antibody for 3 days. AC220 was added to the cultures at days 0, 3 and 7. We assessed CFC numbers present after 2 weeks of culture. Patient samples with low FLT3-AR did not exhibit changes in the numbers of FLT3-ITD CFC (p = 0.735) and FLT3-WT CFC (p = 0.489) in response to Notch inhibition relative to IgG1 control. In contrast, patient samples with high FLT3-AR showed reduction in the number of FLT3-ITD CFC (p=0.019) but the number of FLT3-WT CFC remained unaffected (p=0.874). These results suggest a critical role for Notch in EC-mediated protection in AML with high FLT3-AR.

Conclusion: Our studies suggest that inhibiting Notch signaling may have therapeutic potential for overcoming drug resistance induced by the tumor microenvironment in a subset of AML with high FLT3-AR. We have previously shown that a high FLT3-AR is associated with the presence of FLT3-ITD in the least mature hematopoietic subset (CD34+ CD33- precursors), which is thought to contain leukemic stem cells, and this association is correlated with poorer outcome. Additionally, AML cells that give rise to CFC after long-term co-culture with bone marrow stroma or ECs are derived from the CD34+CD33- AML precursors. Ongoing studies aim to determine whether Notch signaling plays a role in the survival of AML CD34+CD33- cells with the goal of eliminating leukemic stem cells responsible for relapse.