Dynamic clonal progression in xenografts of acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21.

Intrachromosomal amplification of chromosome 21 is a heterogeneous chromosomal rearrangement occurring in 2% of cases of childhood precursor B-cell acute lymphoblastic leukemia. These abnormalities are too complex to engineer faithfully in animal models and are unrepresented in leukemia cell lines. As a resource for future functional and preclinical studies, we have created xenografts from the leukemic blasts of patients with intrachromosomal amplification of chromosome 21 and characterized them by in-vivo and ex-vivo luminescent imaging, flow immunophenotyping, and histological and ultrastructural analyses of bone marrow and the central nervous system. Investigation of up to three generations of xenografts revealed phenotypic evolution, branching genomic architecture and, compared with other B-cell acute lymphoblastic leukemia genetic subtypes, greater clonal diversity of leukemia-initiating cells. In support of intrachromosomal amplification of chromosome 21 as a primary genetic abnormality, it was always retained through generations of xenografts, although we also observed the first example of structural evolution of this rearrangement. Clonal segregation in xenografts revealed convergent evolution of different secondary genomic abnormalities implicating several known tumor suppressor genes and a region, containing the B-cell adaptor, PIK3AP1, and nuclear receptor co-repressor, LCOR, in the progression of B-cell acute lymphoblastic leukemia. Tracking of mutations in patients and derived xenografts provided evidence for co-operation between abnormalities activating the RAS pathway in B-cell acute lymphoblastic leukemia and for their aggressive clonal expansion in the xeno-environment. Bi-allelic loss of the CDKN2A/B locus was recurrently maintained or emergent in xenografts and also strongly selected as RNA sequencing demonstrated a complete absence of reads for genes associated with the deletions.

Post-transcriptional exon shuffling events in humans can be evolutionarily conserved and abundant.

In silico analyses have established that transcripts from some genes can be processed into RNAs with rearranged exon order relative to genomic structure (post-transcriptional exon shuffling, or PTES). Although known to contribute to transcriptome diversity in some species, to date the structure, distribution, abundance, and functional significance of human PTES transcripts remains largely unknown. Here, using high-throughput transcriptome sequencing, we identify 205 putative human PTES products from 176 genes. We validate 72 out of 112 products analyzed using RT-PCR, and identify additional PTES products structurally related to 61% of validated targets. Sequencing of these additional products reveals GT-AG dinucleotides at >95% of the splice junctions, confirming that they are processed by the spliceosome. We show that most PTES transcripts are expressed in a wide variety of human tissues, that they can be polyadenylated, and that some are conserved in mouse. We also show that they can extend into 5' and 3' UTRs, consistent with formation via trans-splicing of independent pre-mRNA molecules. Finally, we use real-time PCR to compare the abundance of PTES exon junctions relative to canonical exon junctions within the transcripts from seven genes. PTES exon junctions are present at <0.01% to >90% of the levels of canonical junctions, with transcripts from MAN1A2, PHC3, TLE4, and CDK13 exhibiting the highest levels. This is the first systematic experimental analysis of PTES in human, and it suggests both that the phenomenon is much more widespread than previously thought and that some PTES transcripts could be functional.

Differential regulation of the androgen receptor by protein phosphatase regulatory subunits.

The Androgen Receptor (AR) is a key molecule in the development, maintenance and progression of prostate cancer (PC). However, the relationship between the AR and co-regulatory proteins that facilitate AR activity in castrate resistant settings remain understudied. Here we show that protein phosphatase 1 regulatory subunits, identified from a phosphatase RNAi screen, direct PP1 catalytic subunits to a varied yet significant response in AR function. As such, we have characterised the PP1ß holoenzyme, myosin phosphatase (MLCP), as a novel ligand independent regulator of the AR. Sustained MLCP activity through down-regulation of the MLCP inhibitory subunit, PPP1R14C, results in impaired AR nuclear translocation, protein stability and transcriptional activity in distinct models of PC progression, culminating in restoration of a non-malignant prostate genotype. Phenotypically, a marked reduction in cell proliferation and migration, characterised by G1 cell cycle arrest is observed, confirming PP1 holoenzyme disruption as a novel treatment approach in PC.