Retroviral Insertional Mutagenesis Reveals Genes That Cooperate with FLT3-ITD in Leukemogenesis.
Authors: Greenberg Kathleen E , Li Li , Huso David , Wolff Linda , Small Donald 
Published: October 2019

Abstract 1960

Poster Board I-983

FLT3 is a receptor tyrosine kinase that plays an important role in hematopoiesis and is one of the most mutated genes in acute myeloid leukemia (AML). The internal tandem duplication (ITD) mutation, which causes autophosphorylation and constitutive activation of the receptor, has been found in up to 35% of AML and 3% of ALL patients. Our lab has created and described a FLT3ITD/wt knock-in mouse with the mutation expressed under the endogenous promoter. These mice develop myeloproliferative disorder (MPD) with a median time to disease of 52 weeks, but do not progress to acute leukemia, indicating that an additional genetic event is required for leukemogenesis. We have used the MOL4070LTR retrovirus in a retroviral insertional mutagenesis (RIM) screen to identify genes that can cooperate with the FLT3/ITD to cause leukemia. FLT3ITD/wt mice were injected with the retrovirus at birth and 78% developed leukemia with a median time to disease of 18 weeks (50% B-ALL/lymphoma, 18% AML, 10% biphenotypic leukemia). By comparison, retrovirus-injected FLT3wt/wt mice developed leukemias with a much longer median time to disease of 69 weeks. The decreased latency in the retrovirally-infected FLT3ITD/wt mice implies that the viral integrations have created mutations that collaborate with FLT3/ITD signaling to generate leukemia. To identify the genes responsible, we cloned the insertion sites using an inverse PCR technique. 343 integration sites were cloned from 25 FLT3ITD/wt mice that developed leukemia, 29 of which were found in two or more mice. These common insertion sites (CIS) are considered putative cooperating genes. The most common insertion, found in 5 mice, involved the Evi1 gene, with the virus inserting approximately 17kb upstream of the gene in three of the mice or approximately 100kb upstream of the gene in the other two mice. All of the mice showed overexpression of Evi1 by quantitative RT-PCR. This is an interesting collaborating gene because its overexpression has been described in AML and predicts a poor prognosis in patients, but mouse models have shown that, like FLT3/ITD, it is not leukemogenic on its own. Though singularly insufficient, together these two mutations may cooperate to cause leukemia. Another putative cooperating gene found in the screen is Ring1a. Insertions upstream of the gene (22kb, 96kb and 209kb) were found in 3 mice displaying myeloid-associated disease, and in all cases caused overexpression of Ring1a, as seen by quantitative RT-PCR. Strong expression of Ring1a, part of the PRC1 polycomb repressor complex, has been observed in several cancer types but not in myeloid leukemia; however, another member of PRC1, Bmi-1, seems to play an extensive role in myeloid leukemia. Integrations near Ring1a have not previously been described in the Retrovirus Tagged Cancer Gene Database (RTCGD mm8), indicating a specific cooperation with FLT3/ITD and a role in the disease. With these studies we have shown that additional mutations cooperate with the FLT3/ITD to cause leukemia and we have identified several possible collaborating genes, including Evi1 and Ring1a.

Disclosures:

No relevant conflicts of interest to declare.