Results from a CRISPR/Cas9 screening study suggest that loss of STK11 promotes progression to leukemia in patients with myeloproliferative neoplasms (MPNs), according to findings presented during the plenary session at the 2020 ASH Annual Meeting. The gene could serve as a potential candidate tumor suppressor gene in patients with post-MPN acute myeloid leukemia (AML), the authors, led by Christian Marinaccio, MSc, from Northwestern University’s Feinberg School of Medicine, in Chicago, Illinois, noted.
Although previous studies have implicated loss of TP53 or JARID2 in MPN progression, by and large the genetic events that lead to conversion to blast phase remain unknown, Mr. Marinaccio explained in his presentation. To better understand determinants of progression, he and researchers performed a focused CRISPR/Cas9 screen to identify genes whose loss promotes disease progression.
In the screen, murine JAK2 V617F bone marrow cells expressing Cas9 were transduced with two lentiviral CRISPR libraries with guide RNAs against 100 tumor suppressors, as well as 50 non-targeting controls. These cells were then used to perform colony formation assay to assess for serial replating and enhanced self-renewal of the JAK2 V617F cells.
Only one gene was targeted by all four guides: STK11. “This gene encodes LKB1, which regulates a number of key cellular pathways including energy utilization by activation of 5’ AMP-activated protein kinase (AMPK),” Mr. Marinaccio reported.
To confirm that loss of STK11 is the event that leads to increased clonogenicity, the investigators collected cells from induced STK11 knockout in murine cells. Consistent with the screening results, only JAK2 V617F cells with Cas9-STK11 single guide RNA showed serial replating.
They also found that activation of JAK/STAT signaling can overcome the requirement for STK11/LKB1 in normal hematopoiesis, suggesting that STK11 loss is a strong driver of malignant transformation in combination with enhanced JAK-STAT signaling.
“We then were interested to assess the mechanism by which loss of STK11 cooperates with enhanced JAK/STAT signaling to promote leukemia, so we performed RNA sequencing on control wild-type and MPL-expressing cells,” Mr. Marinaccio said. This revealed enrichment of a number of pathways related to hypoxia, oxidative phosphorylation, and mitochondrial translation in cells lacking LKB1.
Next, the researchers induced deletion of STK11 in mice that were transplanted with hematopoietic cells that expressed MPL W515L after development of the MPN phenotype to determine whether reduced LKB1 expression was associated with leukemic progression. Both loss of STK11 and LKB1 expression were associated with enhanced bone marrow fibrosis and osteosclerosis.
Then, to determine how loss of STK11 affected human cells, the investigators used CRISPR gene editing to remove STK11 in CD34-positive cells, then transplanted 50,000 cells into mice. Three months after editing, researchers observed increases in circulating human CD45-positive cells in peripheral blood and total human bone marrow cells.
“Consistent with the hypothesis that loss of STK11 facilitates leukemia, we found that its expression was decreased by more than 50% in five of seven paired post-MPN AML patient samples, with two having STK11 levels below 20%,” the authors noted. “We further validated downregulation of LKB1 by immunohistochemistry on paired chronic- and blast-phase MPN specimens and observed little staining in the blast phase specimens.”
Together, these results suggest that JAK-STAT activation overcomes the requirement of STK11/LKB1 in hematopoiesis, allowing for continued growth of STK11-null hematopoietic stem and progenitor cells driven by H1F1-alpha stabilization, Mr. Marinaccio concluded. The finding that pseudohypoxia is present in MPN blast phase cells also could provide avenues of treatment by targeting hypoxia-inducible proteins, he added.
The study authors report no relevant conflicts of interest.
Marinaccio C, Suraneni PK, Celik H, et al. Loss of LKB1/STK11 facilitates leukemic progression of the myeloproliferative neoplasms. Abstract #1. Presented at the 2020 American Society of Hematology Annual Meeting, December 6, 2020.