Cancer scientists reveal the first direct evidence that leukemia development depends on the order in which mutations occur.
Acute myeloid leukemia, or AML, is the most common blood cancer in adults – and one of the most difficult to treat, in part because of the multitude of genes that are mutated in the blood-forming cells of people with the disease.
Researchers at OHSU now have uncovered how two gene mutations act in sequence, like a one-two punch, to trigger AML. The discovery points the way to new targets for therapies to treat AML in people with these mutations.
“This is a subset of patients that do badly,” said Julia Maxson, Ph.D., senior author of the paper in Nature Communications describing the research. “Our hope is that a better biologic understanding will enable the development of more effective therapies.” Maxson is an assistant professor in the OHSU School of Medicine and member of the Knight Cancer Institute.
The need for better therapies is critical. Each year, more than 20,000 Americans are diagnosed with AML and more than 10,000 die from it. The five-year-survival rate is around 30%, versus rates of 70% and higher in other common forms of leukemia.
AML arises in stem cells in the bone marrow that normally mature to become red blood cells, immune system white blood cells, or clot-forming platelets. In people with AML, the intricately regulated output of mature blood cells goes awry. Immature cells multiply rapidly and reach overwhelming numbers in the bone marrow, blood circulation and other organs.
“This new study starts to tell us more about early events in cancer: a gene that helps a white blood cell grow up is broken, and that sets the stage for cancer happening down the road,” said first author Ted Braun, M.D., Ph.D., an instructor of hematology and medical oncology in the OHSU School of Medicine.
Braun, Maxson and colleagues began looking at the role of a gene called CEBPA. It encodes a “master regulator” protein active in prompting stem cells to commit to maturing into different types of blood cells. About three of every ten AML patients with a CEBPA mutation also have a cooperating mutation in a cell signaling receptor gene called CSF3R.
AML patients with dual CSF3R/CEBPA mutations have worse outcomes than those with mutant CEBPA alone. But it has remained unclear how mutations in these two genes interact to drive AML.
To find answers, the OHSU researchers turned to an informative animal model of the disease. They performed bone marrow transplantation to create mice with different combinations of mutated stem cells. These experiments showed that CSF3R and CEBPA cooperate to produce a highly proliferative myeloid leukemia in mice that emulates important aspects of the human disease.
Experiments using the mouse model also showed that mutant CSF3R drives both proliferation and differentiation of maturing myeloid cells. Mutant CEBPA, on the other hand, selectively blocks myeloid differentiation by stopping the work of genetic elements called enhancers. Enhancers help program which genes are turned on and off in stem cells when they commit to becoming a mature differentiated cell type.
‘differentiation-associated enhancers represent a promising new therapeutic target’
For expertise in enhancer biology, Braun and Maxson collaborated with Lucia Carbone, Ph.D., an OHSU associate professor with appointments in the Division of Cardiovascular Medicine, Oregon National Primate Research Center and Department of Molecular and Medical Genetics. “She and members of her lab played a big role in the development and execution of our studies, guiding us to ask better questions,” Maxson said.
Together, the researchers devised a way to change the order in which the mutations occur, and were able to study how mutation order impacts cancer development. In experiments using cells grown in a dish, CSF3R-first mutants proliferated much more slowly than cells with CEBPA-first mutants. Mutation order also effected the pattern of gene activity. CSF3R-first mutants showed signatures of gene activity associated with myeloid differentiation and inflammatory responses. CEBPA-first mutant cells showed stem cell-like gene activity and signatures associated with cell cycle progression.
To really nail down the findings, the researchers developed a way to perform the mutation order experiments in living animals. Mice were transplanted with CSF3R mutant stem cells first, or with CEBPA mutant stem cells first and allowed to recover for 4-weeks post-transplant. After 4-weeks, the second gene mutation was activated.
CEBPA-first mice succumbed to lethal myeloid leukemia with a median survival of 3.5 weeks. The disease in these animals was associated with bone marrow and peripheral blood blasts with immature qualities. In contrast, CSF3R-first mice developed differentiated myeloid cells. After 7 weeks, only one had developed leukemia.
The results show that when CEBPA mutations are introduced after mutations in CSF3R, they are unable to fully block myeloid differentiation. And this impaired ability to block differentiation disrupts the development of AML in a live animal model.
The authors said these findings are the first direct evidence that the order in which oncogenic mutations occur is a major determinant of leukemia development. They said that differentiation-associated enhancers represent a promising new therapeutic target in this poor prognosis subtype of AML.
Myeloid lineage enhancers drive oncogene synergy in CEBPA/CSF3R mutant acute myeloid leukemia by Theodore P. Braun, Mariam Okhovat, Cody Coblentz, Sarah A. Carratt, Amy Foley, Zachary Schonrock, Brittany M. Smith, Kimberly Nevonen, Brett Davis, Brianna Garcia, Dorian LaTocha, Benjamin R. Weeder, Michal R. Grzadkowski, Joey C. Estabrook, Hannah G. Manning, Kevin Watanabe-Smith, Sophia Jeng, Jenny L. Smith, Amanda R. Leonti, Rhonda E. Ries, Shannon McWeeney, Cristina Di Genua, Roy Drissen, Claus Nerlov, Soheil Meshinchi, Lucia Carbone, Brian J. Druker and Julia E. Maxson. Nature Communications (2019)