Multiomic Single Cell Profiling Identifies Drivers of Progression in T-Cell Prolymphocytic Leukemia

Treatments for T-cell prolymphocytic leukemia (T-PLL), while transiently effective in the frontline, have failed to achieve any substantial improvement in the outcomes of this ultra-rare chronic leukemia. Patients with relapsed T-PLL have a dismal prognosis, with a median overall survival (OS) of less than 6 months.

For this study we used samples collected from: 8 patients with untreated T-PLL, 2 with relapsed/refractory (R/R) T-PLL, and 1 from a PDX model of R/R T-PLL. Whole-exome sequencing (WES) was used to elucidate the mutational landscape and single cell T-cell receptor sequencing (scTCRseq), coupled with single cell RNA sequencing (scRNA-seq), was used to generate gene expression and TCR clonality signatures at the single cell level.

The WES revealed a higher frequency of mutations in R/R T-PLL compared to their baseline counterparts. R/R T-PLL samples displayed mutations of CXCR4 (37%; p.S312fs), STAT5B (46%; p.N642H), ATM (82%; p.L2890V), SAMHD1 (92%; p.A565T) and BCOR (p.N1585fs) and deletions in ASXL2 which were absent in treatment naïve T-PLL. In addition, all T-PLL samples harbored recurrent mutations on CXCR4, CD27, JAK1, STAT3, CCND1, CDKN1B, and the epigenetic regulators KMT2C, KMT2B, TET2, and KDM5C. The mutational profiles revealed the genomic complexity of T-PLL. The ubiquitous presence of activating mutations affecting the JAK/STAT axis underscored their importance in driving T-PLL survival. We also studied the diversity of TCR clonality and its relationship with pathogenesis and prognosis in T-PLL. All the samples exhibited dominance of at least one to up to 3 specific CDR3 sequences, revealing the anticipated clonal expansion in T-PLL. Expression of TCRα and TCRβ genes in the dominant clone differed among the samples.Monoclonality was validated as biallelic only when the 2^nd largest transcripts of CDR3α or CDR3β clones were at least 5 times larger than its subceeder. The coupled TCR seq and RNA seq analysis displayed varied degrees of clonal expansions at a single cell resolution across all the patient samples. The T cell subsets were characterized by the expression of various T cell-markers across the cell clusters. In particular, 60% of the samples showed more expression of IL7R and lesser expression CD8A and CD8B, indicating their ability in suppressing the antitumor immunity. CD3E and CD3D were highly expressed across the clusters of T-PLL datasets.Out of 11 datasets, NKG7 and GZMA (cytotoxic T cell markers) were identified in more than 40% of the samples. The augmented levels of anti-apoptosis (BCL-2) and Jak/Stat signaling (JAK1, STAT3) markers were seen across the clusters of T-PLL scRNA seq datasets. The higher expression of PPP1R14B was found in 70% of the samples indicating its probable role in poor prognosis in T-PLL.

In conclusion, T-PLL is characterized by numerous genomic aberrations, presence of 1-3 dominant clones, and gene expression signatures that facilitate survival, and resistance to apoptosis.

Iyer:Astra Zeneca: Research Funding; Merck: Research Funding; Acrotech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Innate: Research Funding; Salarius: Consultancy; IMPaRT.AI: Other: Stock, Founder; Seagen/Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Secura Bio: Membership on an entity's Board of Directors or advisory committees; Yingli: Membership on an entity's Board of Directors or advisory committees, Research Funding; Legend: Research Funding; Crispr: Membership on an entity's Board of Directors or advisory committees, Research Funding; Trillium: Research Funding; Ono: Research Funding; JCO-CCI: Other: Editor. Shukla:Agenus Inc.: Current equity holder in publicly-traded company; Agios Pharmaceuticals: Current equity holder in publicly-traded company; Breakbio Corp.: Current equity holder in publicly-traded company; Bristol-Myers Squibb: Current equity holder in publicly-traded company; Imunon: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Jivanu Therapeutics: Consultancy, Current equity holder in publicly-traded company; Lumos Pharma: Current equity holder in publicly-traded company. Kadia:Incyte: Research Funding; ASTEX: Research Funding; Abbvie: Consultancy, Research Funding; JAZZ: Research Funding; BMS: Consultancy, Research Funding; Rigel: Honoraria; Novartis: Honoraria; Genentech: Consultancy, Research Funding; Regeneron: Research Funding; Amgen: Research Funding; Pfizer: Research Funding; Sellas: Consultancy, Research Funding; Servier: Consultancy; Ascentage: Research Funding; DrenBio: Consultancy, Research Funding; AstraZeneca: Research Funding; Cellenkos: Research Funding. Vega:Caribou: Research Funding; Allogene: Research Funding; Geron Corporation: Research Funding.