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254 Distinct Genetic Pathways Define Leukemia Predisposition Versus Adaptive Clonal Hematopoiesis in Shwachman-Diamond Syndrome

Program: Oral and Poster Abstracts
Type: Oral
Session: 508. Bone Marrow Failure: Advancing Our Biologic Understanding in Inherited and Acquired Bone Marrow Failure Disorders
Hematology Disease Topics & Pathways:
AML, Diseases, Bone Marrow Failure, Biological Processes, Myeloid Malignancies, genomics, Clinically relevant
Saturday, December 5, 2020: 2:00 PM

Alyssa L. Kennedy, MD, PhD1, Kasiani C. Myers, MD2, James Bowman, PhD3*, Christopher J. Gibson, MD4, Gwen M. Muscato5*, Robert Klein1*, Kaitlyn Ballotti5*, Nicholas Camarda1*, Elissa M. Furutani, MD6, Chad E. Harris, MS5*, Shanshan Liu, PhD7*, Ashley Galvin5*, Maggie Malsch, MSN, RN5*, David C. Dale, MD8, John M Gansner, MD, PhD9, Taizo A. Nakano, MD10, Alison A. Bertuch, MD11, Adrianna Vlachos, MD12, Jeff H. Lipton, MD, PhD13, Paul Castillo, MD14*, James Connelly15*, John Edwards, MD16*, Nobuko Hijiya, MD17, Richard Ho, MD18*, Inga Hofmann, MD19, James N Huang, MD20, Sioban B. Keel, MD21, Adam J. Lamble, MD22, Bonnie W Lau, MD, PhD23, Kelly J. Walkovich, MD24, Maxim Norkin, MD25, Wendy Stock, MD26, Steffen Boettcher27*, Christian Brendel, PhD28*, Elliot Stieglitz, MD29, Mark D. Fleming, MD, DPhil30, Stella M. Davies, PhD, MBBS, MRCP31, Edie A. Weller32*, Chris Bahl, PhD3*, Scott L. Carter, PhD33*, Akiko Shimamura, MD, PhD34* and R. Coleman Lindsley, MD, PhD35

1Dana-Farber Cancer Institute, Boston, MA
2Department of Pediatrics, Division of Bone Marrow Transplantation and Immune Deficieny, Cincinnati Children's Hospital, Cincinnati, OH
3Institute for Protein Innovation, Boston, MA
4Department of Medical Oncology, Dana-Farber Cancer Institute, Chestnut Hill, MA
5Boston Children's Hospital, Boston, MA
6Boston Children's Cancer and Blood Disorders Center, Boston Children’s Hospital, Boston, MA
7Division of Hematology and Oncology and Biostatistics and Research Design Center, Institutional Centers for Clinical and Translational Research, Boston Children's Hospital, Boston, MA
8Department of Medicine, University of Washington, Seattle, WA
9Hematology Division, Brigham and Women's Hospital, Newton, MA
10Children's Hospital Colorado, University of Colorado School of Medicine, Denver, CO
11Baylor College of Medicine, Houston, TX
12Feinstein Institute of Medical Research, Cohen Children's Medical Center, Manhasset, NY
13Blood and Marrow Transplant Program, Department of Medical Oncology and Hematology, Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada
14Shand's Children's Hospital, Department of Pediatrics, Division of Pediatric Hematology Oncology, University of Florida, Gainesville, FL
15Department of Pediatrics, Division of Pediatric Hematology Oncology, Vanderbilt University Medical Center, Nashville, TN
16Indiana Blood and Marrow Transplantation, Indianapolis, IN
17Pediatric Hematology Oncology Transplant, Columbia University Irving Medical Center, New York, NY
18Department of Pediatrics, Division of Pediatric Hematology/Oncology, Vanderbilt University Medical Center, Nashville, TN
19Department of Pediatrics, Division of Hematology/Oncology and Bone Marrow Transplantation, University of Wisconsin, Madison, WI
20Division of Pediatric Allergy, Immunology, and Bone Marrow Transplantation, University of California San Francisco, Benioff Children's Hospital, San Francisco, CA
21Department of Medicine, Division of Hematology, Seattle, WA
22Division of Hematology-Oncology, Seattle Children's Hospital, Seattle, WA
23Johns Hopkins University, Grafton, NH
24University of Michigan, Ann Arbor, MI
25University of Florida, Gainesville, FL
26Department of Medicine, University of Chicago, Chicago, IL
27Medical Oncology and Hematology, University and University Hospital Zurich, Zurich, Switzerland
28Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, Harvard Medical School, Boston
29Department of Pediatrics, Division of Hematology-Oncology, UCSF, San Francisco, CA
30Department of Pathology, Boston Children's Hospital, Harvard Medical School, Boston, MA
31Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
32Institutional Centers for Clinical and Translational Research, Biostatistics, Boston Children’s Hospital, Boston, MA
33Dana Farber Cancer Institute, Boston, MA
34Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, Boston, MA
35Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA

Background: Shwachman-Diamond Syndrome (SDS) is a bone marrow failure disorder caused by impaired removal of EIF6 from the nascent 60S ribosome subunit, resulting in defective ribosome assembly. SDS patients have a high risk of myeloid neoplasms (MN) and the prognosis of those that develop MN is poor. Knowledge of the kinetics and functional consequences of somatic mutation acquisition in SDS may offer insight into mechanism of transformation and the potential for therapuetic intervention.

Methods: We performed whole exome sequencing of 45 samples from 30 patients, and validated recurrent somatically mutated genes using targeted sequencing with error suppression in prospectively collected samples from 110 patients in the North American SDS Registry. We correlated mutation status with clinical outcome and performed functional studies to understand the consequence of somatic mutations in SDS.

Results: We detected somatic mutations in 74 of 98 (76%) patients with germline biallelic SBDS mutations (median 2 mutations/patient, range 0-21). We found no mutations in patients with SDS-like disease; those who have clinical features of SDS without disease defining mutations. Of the 83 patients with SDS without a MN diagnosis, 60 (72%) had detectable clonal hematopoiesis (CH), 40 of whom had more than one mutation (median 3, range 1-21). The most frequently mutated genes were EIF6 (60/98, 61%),TP53 (44/98, 45%), PRPF8 (12/98, 12%), and CSNK1A1 (6/98, 6%).

Among SDS patients with TP53 mutated CH, 90.9% (30 of 33) had concurrent EIF6 mutations. To determine whether EIF6 and TP53 mutations occur in the same or different clones, we performed single cell DNA sequencing. Among the 47 clones identified with either EIF6 or TP53 mutations, 24 had a sole EIF6 mutation, and 21 had a sole TP53 mutation, showing that these mutations arise in separate clones.

To study the functional consequences of EIF6 missense mutations, we cloned 7 patient-derived mutations and generated cell lines expressing wild-type or mutant EIF6 cDNA. We found six mutants (I13N, R67W, G69S, P73R, A194T, G196R) reduced levels of EIF6 protein compared with wild type EIF6, despite comparable abundance of mRNA. The most common recurrent mutation, N106S, was found in 20% of patients and, by contrast to others listed above, did not change protein expression. This mutation is located at the EIF6/60S protein interface and disrupted the interaction of N106S-EIF6 with the 60S subunit as measured by polysome profiling followed by western blotting.

To compare the effects of EIF6 versus TP53 somatic mutations in context of SDS deficient translation, we measured ribosome maturation and translation in SDS cells containing shRNAs targeting EIF6 or TP53. EIF6 knockdown ameliorated the SDS defect, reflected by improved ribosome joining (normalization of the 80:60s ratio) and enhanced protein translation (increased O-propargyl-puromycin incorporation), whereas TP53 knockdown had no effect. Knockdown of EIF6 in SDS deficient cells decreased p53 pathway activation as demonstrated by decreased CDKN1A expression.

TP53 mutations were significantly associated with MN diagnosis (p=0.023), but were also common in SDS CH and typically stable over time. To identify the characteristics associated with transformation, we analyzed exomes from 7 patients with TP53 mutated myeloid malignancy for allelic imbalances at the TP53 locus and found that all 7 had biallelic alteration of TP53. Using single cell DNA sequencing from serial samples, we observed that TP53 LOH can precede transformation by several years and can distinguish pre-leukemic clones from indolent clones with monoallelic TP53 alterations. Somatic EIF6 mutations were not found in the leukemic clones. These results suggest early detection of TP53 LOH may distinguish clones with leukemic potential.

Conclusions: In SDS, impairment of ribosome maturation drives selection of clones with somatic EIF6 or TP53 mutations. EIF6 mutations promote competitive fitness by rescuing the SDS ribosome defect and decreasing p53 pathway activation, and do not contribute to malignant transformation. TP53 mutations decrease checkpoint activation without affecting ribosome assembly. These results provide genetic evidence that germline SBDS deficiency causes a global, disease-specific HSC fitness constraint that drives parallel development of somatic CH and provides a mechanistic rationale for clinical surveillance.

Disclosures: Dale: Emendo BioTherapeutics: Consultancy; X4 Pharmaceuticals: Research Funding; X4 Pharmaceuticals: Honoraria. Gansner: Alnylam Pharmaceuticals: Current Employment, Current equity holder in private company. Edwards: Jazz Pharmaceuticals: Consultancy, Honoraria. Fleming: DISC Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lindsley: Bluebird Bio: Consultancy; Takeda Pharmaceuticals: Consultancy; MedImmune: Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding.

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