Resistance to the graft-versus-leukemia (GvL) effect remains the major barrier to successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) for aggressive hematologic malignancies. The basis of GvL resistance for advanced lymphoid malignancies remains incompletely understood. We hypothesized that for patients with chronic lymphocytic leukemia (CLL) treated with allo-HSCT, leukemic cell-intrinsic features shape GvL outcomes by directing the evolutionary trajectories of CLL cells. Integrated genetic, transcriptomic and epigenetic analyses of CLL cells from 10 patients revealed that the clinical kinetics of post-HSCT relapse are shaped by distinct molecular dynamics and suggest that the selection pressures of the GvL bottleneck are unlike those imposed by chemotherapy. No selective advantage for HLA loss was observed, even when present in pre-transplant subpopulations. Regardless of post-transplant relapse kinetics, gain of stem cell modules was a common signature associated with leukemia relapse. These data elucidate the biological pathways that underlie GvL resistance and post-transplant relapse.### Competing Interest StatementC.J.W. is a co-founder of Neon Therapeutics and member of its scientific advisory board. C.J.W., G.G., D.N. and T.J.K. receive research funding from Pharmacyclics. T.J.K. has received research funding and/or has served as an advisor to Ascerta/AstraZeneca, Celgene, Genentech/Roche, Gilead, Janssen, Loxo Oncology, Octernal Therapeutics, Pharmacyclics/AbbVie, TG Therapeutics, VelosBio, and Verastem. Cirmtuzumab was developed by T.J.K. and licensed by the University of California to Oncternal Therapeutics, Inc., which has provided stock/options to the university and T.J.K. G.G. receives research funds from IBM. G.G. is an inventor of several bioinformatics-related patents, including patents related to MuTect and ABSOLUTE. J.R.B. is a consultant for Abbvie, Acerta, Beigene, Genentech/Roche, Gilead, Juno/Celgene, Kite, Loxo, Novartis, Pfizer, Pharmacyclics, Sunesis, TG Therapeutics and Verastem; received honoraria from Janssen and Teva; received research funding from Gilead, Loxo, Sun and Verastem; and served on data safety monitoring committees for Morphosys and Invectys. S.A.S. previously advised and has received consulting fees from Neon Therapeutics. S.A.S. reported nonfinancial support from Bristol-Myers Squibb, and equity in Agenus Inc., Agios Pharmaceuticals, Breakbio Corp., Bristol-Myers Squibb and NewLink Genetics, outside the submitted work. D.N. reports stock ownership in Madrigal Pharmaceuticals. J.R. receives research funding from Amgen, Equillium and Kite/Gilead and serves on Data Safety Monitoring Committees for AvroBio and Scientific Advisory Boards for LifeVault Bio, Rheos Medicines, Talaris Therapeutics and TScan Therapeutics. P.V.K. serves on the Scientific Advisory Board to Celsius Therapeutics Inc. The remaining authors declare no competing financial interests.

Distinct evolutionary paths in chronic lymphocytic leukemia during resistance to graft-versus-leukemia