Abrogation of a Histone Chaperone Pathway Mitigates Inflammation-Driven AML Progression

Background: Genetic heterogeneity makes clinical interventions challenging for acute myeloid leukemia (AML) patients. Identifying and targeting microenvironment-driven pathways that are active across AML genetic subtypes should allow the development of more broadly effective therapies. Previously, we have shown that AML microenvironment is rich in proinflammatory cytokine interleukin-1β (IL-1β) and significantly promotes the growth of AML progenitors while suppressing healthy progenitors. To elucidate this paradoxical effect, we performed transcriptome (RNA-seq) analysis from IL-1β-stimulated CD34+ AML and normal progenitors and found that ASF1B (anti-silencing function-1B) is one of the most differentially expressed genes. ASF1B is a histone chaperone, which recruits H3-H4 histones onto the replication fork during S-phase. This process is regulated by tousled-like kinase 1 and 2 (TLKs). TLKs and ASF1B are overexpressed in multiple solid tumors and associated with poor prognosis. However, their functional roles in hematopoiesis and inflammation-driven leukemia are unexplored. Here, we reveal a novel molecular mechanism that IL-1β promotes leukemia progression by activating the TLK-ASF1B pathway.

Methods and Results: We first confirmed that IL-1β stimulation upregulates ASF1B expression at both mRNA and protein levels in FLT3-ITD, MLL-ENL, and NPM1 positive primary AML samples. ASF1B upregulation is abolished upon treatment with a p38MAPK inhibitor, further suggesting that ASF1B is downstream of IL-1β/p38 signaling. Next, we stably knocked down ASF1B in an AML cell line MOLM-14 using doxycycline-inducible shRNA. ASF1B-depleted AML cells exhibited reduced cell viability, inhibited growth, blocked cell cycle progression, and impaired colony formation ability. We xenografted shASF1B expressing AML cells into NSG mice and induced knockdown in vivo. Flow cytometry analysis of bone marrow cells 3 weeks post-engraftment showed 80 percent reduction in leukemia burden following ASF1B silencing compared to controls.

To determine whether upregulation of ASF1B contributes to IL-1β-driven leukemic growth, we overexpressed ASF1B with MLL-ENL oncogene in a murine bone marrow transplantation model. We found that daily IL-1β exposure accelerated leukemia progression compared to vehicle-treated group (median survival = 64 vs. 85 days, p<0.05), and this effect was phenocopied by overexpression of ASF1B (median survival = 62 vs. 85 days, p<0.05). Conversely, heterozygous and complete Asf1b deletion in the MLL-ENL AML model delayed the leukemia progression compared to wildtype mice. Furthermore, Asf1b deletion attenuated IL-1β-mediated AML progression compared to wildtype controls (median survival = 63 vs. 47 days, p <0.01). Immunophenotyping of Asf1b-deficient mice using flow cytometry suggested that ASF1B is dispensable for normal hematopoiesis. Together, these data suggested that targeting of the ASF1B pathway may spare healthy cells. Additionally, we found that TLK2 which regulates ASF1B activation, is also upregulated in AML progenitors compared to healthy cells at the baseline levels and upon IL-1β stimulation. We next knocked down TLKs in human AML cells and observed a similar pattern with growth arrest, higher replication stress and DNA damage response. Finally, we generated Vav-Cre+ Tlk2 mice allowing Tlk2 deletion only in hematopoietic cells and found that Tlk2 deletion prolongs survival of leukemic mice in a dose dependent manner with and without IL-1β stimulation.

Conclusions: We demonstrate that increased TLK-ASF1B expression promotes survival of AML cells. We also provide the first in vitro and in vivo evidence that the TLK-ASF1B pathway plays a critical role in potentiating IL-1β-dependent AML growth. Therefore, we establish TLK-ASF1B pathway as a novel route for therapeutic strategy to suppress inflammation-driven growth in various AML genetic subtypes.