Genetic Analysis of B-Cell Acute Lymphoblastic Leukemia Dissemination to the Central Nervous System Identifies Clonal Selection and Therapeutic Vulnerability
Authors: Vanner Robert , Dobson Stephanie M. , Grandal Ildiko , Gan Olga I. , McLeod Jessica , Kennedy James , Voisin Veronique , Guidos Cynthia , Danska Jayne S. , Waanders Esme , Minden Mark D. , Mullighan Charles G. , Dick John E. 
Published: October 2019

Without prophylactic therapy, B-cell Acute Lymphoblastic Leukemia (B-ALL) spreads to the leptomeninges of the central nervous system (CNS) in up to 70% of patients. CNS involvement is more common in certain high risk B-ALL subgroups, including patients with KMT2A (MLL)-translocations, and disease relapse in the CNS carries a poor prognosis. The genetic determinants and biology of B-ALL dissemination to the CNS are poorly defined and therefore therapies targeting the drivers of CNS disease are lacking. Whereas B-ALL exhibits significant subclonal diversity that contributes to functional heterogeneity and disease relapse, recent reports suggest similar clonal composition of bone marrow (BM) and CNS disease, with the potential for CNS dissemination being a universal property of B-ALL cells (Williams et al. 2016, Bartram et al. 2018). Furthermore, functional studies of leptomeningeal disease have focused on the invasion of B-ALL cells into the CNS but limited studies have addressed the selection of genetic clones with the ability to grow within the subarachnoid space.

To better define the evolutionary history and biology of leptomeningeal B-ALL we performed targeted DNA, SNP copy number, RNA sequencing, and functional analysis on cells isolated from matched BM and CNS tissue of patient derived xenografts (PDX) generated from a cohort of paired diagnosis and relapse samples from 14 pediatric and adult B-ALL patients of varying cytogenetics. The majority of primary patient samples yielded CNS disease 20 weeks after intrafemoral injection into NSG mice. CNS disease burden was higher in PDXs derived from relapsed B-ALL samples. Human B-ALL cells isolated from the CNS of PDXs retained competence to repopulate disease in the BM, spleen, and CNS upon serial transplantation. Targeted DNA sequencing results analyzed using a Bayesian clustering method revealed different genetic clonal composition between matched BM and CNS cells in approximately half of the xenografts. PDXs from relapse samples were more likely to show genetic discordance between the BM and CNS. Copy number analysis also confirmed frequent genetic discordance between cells isolated from the BM and CNS from individual PDXs. Interestingly, in one patient all PDXs generated from the relapse sample displayed chromosome 6p and 17p hemi-deletions that were unique to the CNS. In total, PDXs from four patients showed recurrent enrichment of specific lesions in CNS-engrafting cells, suggesting that transit to and/or survival within the subarachnoid space can be the product of selection for genetic clones with increased CNS tropism.

RNA-seq of matched BM and CNS cells derived from 45 of the primary PDXs demonstrated that CNS-isolated cells were transcriptionally distinct from their matched BM. These differences were most pronounced in samples from patients with MLL-AF4 translocations, whose CNS isolated cells grouped together in multi-dimensional scaling. Using GSEA, the most highly CNS-enriched gene sets in MLL samples were related to mRNA translation initiation and polypeptide elongation. Translation-related gene sets are similarly enriched in the blasts of MLL B-ALL patients with CNS disease in the COG 9906 study. CNS-isolated cells from PDXs of MLL patients exhibited altered rates of protein synthesis compared to matched BM-isolated cells. Treatment of PDXs with the clinically-approved translational inhibitor omacetaxine mepesuccinate (OMA) effectively decreased rates of translation in CNS-engrafting cells. Moreover, OMA reduced leukemia burden nearly 4-fold in PDXs bearing established CNS infiltration generated from two MLL patients.

Our data represent an advance in the understanding of B-ALL CNS disease. We present a rich resource of genomic and transcriptomic data from xenografts spanning multiple B-ALL subgroups across diagnosis and relapse and have identified selection for genetically and biologically distinct clones in the CNS, contrary to the current model. Furthermore, we demonstrate that in MLL patients, dysregulation of protein synthesis occurs at CNS dissemination and targeting this process is a novel therapeutic paradigm that may benefit patients with CNS disease.

Disclosures

Mullighan: Cancer Prevention and Research Institute of Texas: Consultancy; Pfizer: Honoraria, Research Funding, Speakers Bureau; Loxo Oncology: Research Funding; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding.