Grant Challen, Ph.D.
Project Summary:
Hematopoietic stem cells (HSCs) reside in the bone marrow and are responsible for the lifetime generation of all the cells of the blood system. Blood cancers such as acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) arise from genetic mutations in HSCs which alter how the cells divide and make mature blood cells. New advances in genome sequencing have revealed the spectrum of mutations present in AML and MDS patients and highlighted how these diseases develop. Most of these cancers have relatively few mutations, which are acquired in a stepwise procession over a long period of time. The first mutation acquired (the “founding” or “initiating” mutation) generally acts to stop the HSCs from turning into mature blood cells, which would lead the mutant cell to reproduce itself and take over in the bone marrow. This process is called clonal expansion. Many founding mutations affect genes involved in controlling DNA modifications called epigenetic marks, with the two most common being the enzymes DNMT3A and TET2, which regulate the epigenetic mark of DNA methylation. In the next step to cancer, a mutant cell acquires a second genetic mutation that increases proliferation. But the rate of this progression is highly variable, and the timeframe for acquiring these cancer-causing mutations in the correct order appears to be on the scale of decades. We know this because mutations in DNMT3A and TET2 are commonly found in the blood of healthy elderly people who have no signs of disease. This is known as clonal hematopoiesis of indeterminate potential (CHIP). An individual with CHIP has a 100-fold increased lifetime risk of developing a blood cancer. However, any way to slow the growth of these mutant cells during the CHIP phase will reduce that individual’s lifetime risk for blood cancer and represent a breakthrough in cancer prevention.
Despite advances in genomics, cure rates for AML and MDS patients have not substantially improved in the last 40 years. Rather than focus on new treatments for mature disease, which may only extend a patient’s lifespan by a few months, we propose to eliminate the HSCs carrying cancer-causing mutations before they have a chance to acquire secondary mutations. The long timescale required for the evolution of these diseases presents a unique opportunity for cancer prevention. Using sensitive deep-sequencing techniques, we can accurately identify elderly individuals who carry blood cells with DNMT3A or TET2 mutations. The goal of this project is to develop a way to weed out mutant HSCs while sparing the normal healthy cells by targeting pathways only the mutant cells require for their propagation (essentially, a molecular weedkiller). This is a method of cancer prevention by reducing the chance of an “at-risk” CHIP individual progressing to AML or MDS.
Progress Report
A Platform to Screen for Genetic Determinants of Pre-Malignant HSC Self-Renewal: We have used conditional knockout mice to show how loss-of-function mutations in Dnmt3a and Tet2 (henceforth referred to as Dnmt3aKO and Tet2KO respectively) cause a pre-malignant phenotype of HSC clonal expansion with disproportionate contribution to hematopoiesis. Loss of Dnmt3a and Tet2 in HSCs enhance self-renewal in HSCs, analogous to humans with CHIP. We have developed a protocol in the lab exploiting the enhanced self-renewal of Dnmt3aKO and Tet2KO HSCs to generate cell lines from individual clones, which can be grown as suspension cells in liquid media. These “Epigenetic-Mutant Pre-Malignant HSC lines” (EMPs) provided us with a platform for conducting genetic screens in a cell type that more faithfully represents the cells we ultimately hope to target in vivo. We used these EMPs to perform an initial CRISPR/Cas9 negative selection discovery screen to using a guide RNA (gRNA) library targeting gene involved in epigenetic regulation (all of which are in principle “druggable”). This provided our initial list of candidate targets.
We performed a complimentary CRISPR/Cas9 negative selection screen in a mouse hematopoietic progenitor cell line (32D cells) engineered with Dnmt3a and Tet2 mutations. This was done to include a “wild-type” genotype comparator since normal HSCs from mice cannot be propagated for our screen in primary cells. This data set has been used as a validation tool to refine our candidate gene list from the screen in premalignant HSCs. Our finalized candidate gene list for Dnmt3a dependencies = Brd2, Zmynd8, Jmjd1c, Mll5, Prdm2, Prdm8, Prdm9, Set1a, and Atf2. Our finalized candidate gene list for Tet2 dependencies = Brd2, Zmynd8, Kdm6b, and Mll5. These assays were finalized at the beginning of the funding period.
