Elena Sotillo

Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy

UNLABELLED The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. SIGNIFICANCE CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.

Shielding the messenger (RNA): microRNA-based anticancer therapies

It has been a decade since scientists realized that microRNAs (miRNAs) are not an oddity invented by worms to regulate gene expression at post-transcriptional levels. Rather, many of these 21-22-nucleotide-short RNAs exist in invertebrates and vertebrates alike and some of them are in fact highly conserved. miRNAs are now recognized as an important class of non-coding small RNAs that inhibit gene expression by targeting mRNA stability and translation. In the last ten years, our knowledge of the miRNAs world was expanding at vertiginous speed, propelled by the development of computational engines for miRNA identification and target prediction, biochemical tools and techniques to modulate miRNA activity, and last but not least, the emergence of miRNA-centric animal models. One important conclusion that has emerged from this effort is that many microRNAs and their cognate targets are strongly implicated in cancer, either as oncogenes or tumor and metastasis suppressors. In this review we will discuss the diverse role that miRNAs play in cancer initiation and progression and also the tools with which miRNA expression could be corrected in vivo. While the idea of targeting microRNAs towards therapeutic ends is getting considerable traction, basic, translational, and clinical research done in the next few years will tell whether this promise is well-founded.

Repeated loss of target surface antigen after immunotherapy in primary mediastinal large B cell lymphoma.

Washington, District of Columbia; Hematology Oncology, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts; Medicine, Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts; Phoenicia Biosciences, Weston, Massachusetts Conflict of interest: S. Perrine: Inventor on patents related to this work. Contract grant sponsor: NIH; Contract grant numbers: 1P50 HL-118006, R01 DK-52962, R41 HL-108516, R42 HL-110727. *Correspondence to: Susan Perrine, MD, Hemoglobinopathy Thalassemia Research Unit, Boston University School of Medicine, 72 East Concord Street, L909, Boston, MA 02118. E-mail: sperrine@bu.edu Received for publication: 27 September 2016; Revised: 13 October 2016; Accepted: 17 October 2016 Published online: 20 October 2016 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ajh.24590

The long reach of noncoding RNAs.

Transcription of genomic loci containing protein-coding genes often yields not only cognate mRNAs but also assorted noncoding RNAs (ncRNAs), which typically map in the vicinity of transcription start sites. A new study shows that far from being random byproducts of gene expression, many long ncRNAs (lncRNAs) are synthesized in a coordinate fashion and control important cellular processes, such as survival in the face of DNA damage.

Escape from Cellular Quiescence

Quiescent: From Latin quies, referring to a state of being at rest, dormant, inactive, quiet, still (Merriam-Webster, 2009, Online Dictionary: http://www.merriam-webster.com/dictionary/quiescent). This term refers to a state of dormancy as opposed to a proliferative state. However, quiescent cells are in any other regard metabolically active. In many tissues with relative fast cell renewal rates the primary function of a small group of undifferentiated cells is limited to self-renewal (stem cells). These cells remain quiescent most of the time dividing only occasionally. In other tissues, key cell types perform fundamental tissue functions while remaining quiescent. Both stem cells and cells from tissues that renew via simple duplication can remain quiescent for long periods of time while retaining the capacity to re-enter the cell cycle. This chapter will discuss the mechanisms emerging as responsible for the maintenance of quiescence as well as those pathways that mediate quiescence entry and exit. We will also review signaling pathways deregulated during infection by Simian Virus 40 (SV40) and oncogenic transformation, which result in unscheduled exit from quiescence into the cell cycle, with focus on SV40 small t antigen.

Abstract 3143: Alternative splicing of CD19 mRNA in leukemias escaping CART-19 immunotherapy eliminates the cognate epitope and contributes to treatment failure

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA CD19 is expressed on most B-cell neoplasms, including acute lymphoblastic leukemias (B-ALL). Chimeric antigen receptor (CAR)-armed T cells targeting CD19 (CART-19) yield complete remission rate of 70-90% in B-ALL patients; the bispecific anti-CD19/CD3 antibody blinatumomab also shows clinical efficacy. Yet relapses due to the apparent CD19 loss have been observed following both therapies. Here we investigated the molecular mechanisms of epitope loss using five B-ALL samples obtained after CD19-directed therapy. These samples were matched, when available, with CD19-positive pre-treatment leukemias. Although the CD19 epitope recognized by CART-19 was undetectable in relapsed specimens, in all cases the CD19 genomic sequence was retained and the mRNA levels were only slightly reduced, suggesting regulation at the level of transcript processing. Indeed, using RT-PCR and RNA-Seq we detected several alternatively spliced CD19 mRNA species lacking exons 5-6 and/or exon 2 (Δex2), which encodes the cognate CART-19 epitope. In the sets of matched samples, the relative abundance of the Δex2/5-6 mRNA isoforms was increased post-treatment, and accordingly truncated CD19 protein variants were detectable using antibodies recognizing alternative epitopes. In one relapsed leukemia with a nonsense mutation in exon 2 of CD19, Δex2 was the only expressed isoform. To determine the contribution of alternative splicing to immune escape, we introduced CD19-expressing retroviruses into murine neoplastic B-cells, which had lost endogenous CD19 expression following silencing of its main transcriptional regulator Pax5. While skipping of exon 2 made the resultant protein invisible to CART-19, it partly rescued the defects in cell proliferation associated with CD19 loss. Collectively, these data suggest the existence of a novel mechanism of resistance to immunotherapy, based not on mutations in the coding sequence but rather on selection for alternatively spliced target protein isoforms (e.g., CD19 Δex2). Our findings could lead to the development of additional CD19-targeting CARTs for treating relapsed patients. Citation Format: Elena Sotillo, David Barrett, Aseb Bagashev, Kathryn Black, Caludia Lanauze, Derek Oldridge, Robyn Sussman, Colleen Harrington, Elaine Y. Chung, Ted J. Hofmann, Shannon L. Maude, Nicole M. Martinez, Pichai Raman, Marco Ruella, David Allman, Elad Jacoby, Terry Fry, Yoseph Barash, Kristen W. Lynch, Crystal Mackall, John Maris, Stephen A. Grupp, Andrei Thomas-Tikhonenko. Alternative splicing of CD19 mRNA in leukemias escaping CART-19 immunotherapy eliminates the cognate epitope and contributes to treatment failure. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3143. doi:10.1158/1538-7445.AM2015-3143