Leveraging Pathway-Interference to Overcome Drug-Resistance in Acute Lymphoblastic Leukemia

Background: The concept of multi-step carcinogenesis (Fearon and Vogelstein 1990) suggests that acquisition of mutations in addition to an existing set of mutations invariably accelerates tumor-progression. In colorectal cancer and many other cancer types, activation of multiple distinct oncogenic pathways is required for the development of invasive cancer. Here, we examined this paradigm for genetic lesions in B-ALL and 13 other cancer types. Bioinformatic approaches: To broadly study how oncogenic drivers across multiple signaling pathways interact, we developed a bioinformatic platform to map interactions between genetic lesions that cause oncogenic activation of eight oncogenic pathways, including PI3K, STAT5, NF-κB, Hippo, Notch, WNT, RAS-ERK and TGFβ-Smad pathways. Plotting of interaction scores between 56 pathway pairs in a matrix for 14 cancer types revealed that 12 of the 14 cancer types showed a pattern of globally synergistic pathway interactions, consistent with the Fearon and Vogelstein model of cooperation of multiple pathways to drive malignant transformation. Strikingly, B-ALL and gliomas showed the opposite behavior with largely antagonistic pathway interactions. Results. Unlike the vast majority of cancer types, B-ALL and gliomas are driven by one principal oncogenic pathway at a time. While the reasons for negative pathway interactions in glioma are unknown, we focused on functional analyses on pathway interference patterns in 1,148 cases of B-ALL. Genetic lesions leading to STAT5- or ERK-pathway activation are frequently found in B-ALL. Interestingly, activating lesions of both pathways co-occurred in only 3% of the cases studied, suggesting that co-activation of STAT5- and ERK-occurs much less frequently than expected by chance (odds ratio 0.13, P=2e-16, Figure, left). Unbiased interaction mapping analyses of mutational co-occurrence indicated strong negative selection for dual activation of both STAT5- and ERK-pathways (Figure, middle). Importantly this inter-pathway aversion is much stronger than intra-pathway effects, reflecting incompatibility rather than redundancy. Even in rare cases of co-occurrence in the same sample, single-cell mutation and phosphoprotein analyses revealed that STAT5- and ERK-activating mutations were mutually exclusive and reflected two competing clones. STAT5- and ERK-pathways engage conflicting transcriptional and biochemical programs, resulting in "friction", when both pathways are concurrently activated. In agreement with pathway interference, we demonstrated that Cre-mediated deletion of divergent pathway components - Erk2 fl/fl in a STAT5-driven model of B-ALL and Stat5 fl/fl in an ERK-driven B-ALL model - dramatically accelerated initiation of fatal leukemia in vivo. While Cre-mediated deletion of divergent pathway components precipitated leukemia-initiation, these findings suggest that reactivation of divergent signaling pathways represents a powerful barrier against malignant transformation. Interestingly, our preclinical studies suggested that pharmacological reactivation of divergent (suppressed) pathways can be leveraged for therapeutic benefit: The DUSP6 small molecule inhibitor BCI-215 functions as powerful activator of ERK and suppresses STAT5-phosphorylation, i.e. the principal pathway in STAT5-driven B-ALL (Figure, right). Likewise, DPH, a small molecule STAT5-agonist interferes with ERK-phosphorylation, the principal oncogenic driver in RAS-pathway B-ALL (Figure, right). Both BCI-215 and DPH significantly prolonged overall survival of NSG mice transplanted with refractory STAT5- and ERK-driven B-ALL PDX, respectively. Conclusions: We propose that a diverse spectrum of signaling input reflects interactions of normal cells with their environment, while convergence on one centralized pathway is a hallmark of cancer. Tracking early stages of leukemia-initiation, we identified convergence on one principal oncogenic driver and inactivation of diverging pathways as an early critical step. Pharmacological reactivation of divergent signaling pathways to subvert transformation was achievable by STAT5- and ERK-agonists. Proof-of-concept studies in patient-derived B-ALL cells revealed that pharmacological reactivation of suppressed divergent circuits can be leveraged as a previously unrecognized strategy to overcome drug-resistance. Figure 1 Figure 1. Disclosures Izraeli: Roche: Consultancy, Speakers Bureau; Bayer: Speakers Bureau; sightDx: Consultancy. Weinstock: ASELL: Consultancy; SecuraBio: Consultancy; Bantam: Consultancy; AstraZeneca: Consultancy; Abcuro: Research Funding; Verastem: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Travera: Other: Founder/Equity; Ajax: Other: Founder/Equity.

Genome-Wide Assessment Characteristics of Genes Overlapping Copy Number Variation Regions in Duroc Purebred Population

Copy number variations (CNVs) are important structural variations that can cause significant phenotypic diversity. Reliable CNVs mapping can be achieved by identification of CNVs from different genetic backgrounds. Investigations on the characteristics of overlapping between CNV regions (CNVRs) and protein-coding genes (CNV genes) or miRNAs (CNV-miRNAs) can reveal the potential mechanisms of their regulation. In this study, we used 50 K SNP arrays to detect CNVs in Duroc purebred pig. A total number of 211 CNVRs were detected with a total length of 118.48 Mb, accounting for 5.23% of the autosomal genome sequence. Of these CNVRs, 32 were gains, 175 losses, and four contained both types (loss and gain within the same region). The CNVRs we detected were non-randomly distributed in the swine genome and were significantly enriched in the segmental duplication and gene density region. Additionally, these CNVRs were overlapping with 1,096 protein-coding genes (CNV-genes), and 39 miRNAs (CNV-miRNAs), respectively. The CNV-genes were enriched in terms of dosage-sensitive gene list. The expression of the CNV genes was significantly higher than that of the non-CNV genes in the adult Duroc prostate. Of all detected CNV genes, 22.99% genes were tissue-specific (TSI > 0.9). Strong negative selection had been underway in the CNV-genes as the ones that were located entirely within the loss CNVRs appeared to be evolving rapidly as determined by the median dN plus dS values. Non-CNV genes tended to be miRNA target than CNV-genes. Furthermore, CNV-miRNAs tended to target more genes compared to non-CNV-miRNAs, and a combination of two CNV-miRNAs preferentially synergistically regulated the same target genes. We also focused our efforts on examining CNV genes and CNV-miRNAs functions, which were also involved in the lipid metabolism, including DGAT1, DGAT2, MOGAT2, miR143, miR335, and miRLET7. Further molecular experiments and independent large studies are needed to confirm our findings.

Complete Mitogenome of Endangered and Endemic Nicobar Treeshrew (Tupaia Nicobarica) and Comparison with Other Scandentians

The Nicobar treeshrew (Tupaia nicobarica) is an endangered smaller mammal endemic to the Nicobar Island of the Andaman Sea, India regarded as an alternative experimental animal model in biomedical research. The present study aimed to assemble the first mitochondrial genome of T. nicobarica to elucidate evolutionary relationship. The structure and variation of the novel mitochondrial genome were analyzed and compared with other Scandentians. The complete mitogenome (17,164 bp) encodes 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNAs), two ribosomal RNA (rRNAs), and one control region (CR). Most of the genes were encoded on majority strand, except nad6 and eight tRNAs. The nonsynonymous/synonymous ratio in all PCGs indicates strong negative selection among all Tupaiidae species. The comparative study of CRs revealed the occurrence of tandem repeats (CGTACA) found in T. nicobarica. The phylogenetic analyses (ML and BA) showed distinct clustering of T. nicobarica with high branch supports and depict a substantial divergence time (11.4 to 18.8 MYR) from the ancestor lineage of Tupaiidae. The 16S rRNA dataset corroborates the taxonomic rank of two subspecies of T. nicobarica from the Great and Little Nicobar Islands. The present study suggests the assembly of whole-genome to improve the understanding of evolutionary relationships of treeshrews and its implication in biomedical research.

Genome-Wide Identification and Analysis of the Metallothionein Genes in Oryza Genus

Metallothionein (MT) proteins are low molecular mass, cysteine-rich, and metal-binding proteins that play an important role in maintaining metal homeostasis and stress response. However, the evolutionary relationships and functional differentiation of MT in the Oryza genus remain unclear. Here we identified 53 MT genes from six Oryza genera, including O. sativa ssp. japonica, O. rufipogon, O. sativa ssp. indica, O. nivara, O. glumaepatula, and O. barthii. The MT genes were clustered into four groups based on phylogenetic analysis. MT genes are unevenly distributed on chromosomes; almost half of the MT genes were clustered on chromosome 12, which may result from a fragment duplication containing the MT genes on chromosome 12. Five pairs of segmental duplication events and ten pairs of tandem duplication events were found in the rice MT family. The Ka/Ks values of the fifteen duplicated MT genes indicated that the duplicated MT genes were under a strong negative selection during evolution. Next, combining the promoter activity assay with gene expression analysis revealed different expression patterns of MT genes. In addition, the expression of OsMT genes was induced under different stresses, including NaCl, CdCl2, ABA, and MeJ treatments. Additionally, we found that OsMT genes were mainly located in chloroplasts. These results imply that OsMT genes play different roles in response to these stresses. All results provide important insights into the evolution of the MT gene family in the Oryza genus, and will be helpful to further study the function of MT genes.

Validation of predicted anonymous proteins simply using Fisher’s exact test

MotivationGiven its increasing efficiency, accuracy, and decreasing cost, genomes sequencing has become the primary (and often the sole) experimental method to characterize newly discovered organisms, in particular from the microbial world (bacteria, archaea, viruses). This generates an ever increasing number of predicted proteins the existence of which is unwarranted, in particular when they do not share significant similarity with proteins of model organisms. As a last resort, the computation of the selection pressure from pairwise alignments of the corresponding “Open Reading Frames” (ORFs) can validate their existences. However, this approach is error-prone, as not usually associated with a significance test.ResultsWe introduce the use of the straightforward Fisher’s exact test as a post processing of the results provided by the popular CODEML pairwise sequence comparison software. The respective rates of nucleotide changes at the non-synonymous vs. synonymous position (as determined by CODEML), are turned into entries into a 2×2 contingency table, the probability of which is computed under the Null hypothesis that they should not behave differently (i.e. the ORFs do not encode real proteins). I show that strong negative selection pressures do not always provide a significant argument in favor of the existence of proteins.

Phylogenetic and diversity analyses revealed that leek yellow stripe virus population consists of two types: S and N

Leek yellow stripe virus (LYSV, Potyvirus), a pathogen affecting Allium spp. worldwide, has been suspected to consist of two types: S and N, based on genetic and host species differences. In this study, phylogenetic and evolutionary analyses were performed to P1 and CP regions of genome of global LYSV isolates to clarify the variation among members of S-type and N-type. Constructed phylogenetic trees clearly divided isolates into S-type and N-type, with N-type was further split into L and N groups, according to hosts. Significant nucleotide (nt) and amino acids (aa) sequence variation were observed on full ORF, P1, HC-Pro, P3, VPg coding regions. The dN/dS values of P1 and CP confirmed that both genes are under strong negative selection pressure. Neutrality tests on Eastern Asian isolates argued that ancestor of current LYSV isolates may had evolved with garlic while they were in Asia before spread to other world regions through garlic propagative materials. The genetic differentiation and gene flow analysis showed that there was very frequent gene flow from S-type to L and N groups and these phylogroups differentiated from each other over time. Host differences, substantial nt and aa variation, inconsistent serological test results, and phylogenetic and diversity analyses results highly suggested that LYSV can be separated into two types: S and N.

The haplolethality paradox of the wupA gene in Drosophila

Haplolethals (HL) are regions of diploid genomes that in one dose are fatal for the organism. Their biological meaning is obscure because heterozygous loss-of-function mutations result in dominant lethality (DL) and, consequently, should be under strong negative selection. We report an in depth study of the HL associated to the gene wings up A (wupA). It encodes 13 transcripts (A-M) that yield 11 protein isoforms (A-K) of Troponin I (TnI). They are functionally diverse in their control of muscle contraction, cell polarity and cell proliferation. Isoform K transfers to the nucleus where it increases transcription of the cell proliferation related genes CDK2, CDK4, Rap and Rab5. The nuclear translocation of isoform K is prevented by the co-expression of A or B isoforms, which illustrates isoform interactions. The corresponding DL mutations are, either DNA rearrangements clustered towards the gene 3’ end, thus affecting the genomic organization of all transcripts, or CRISPR-induced mutations in one of the two ATG sites which eliminate a subset of wupA products. The joint elimination of isoforms C, F, G and H, however, do not cause DL phenotypes. Genetically driven expression of single isoforms rescue neither DL nor any of the mutants known in the gene, suggesting that normal function requires properly regulated expression of specific combinations, rather than single, TnI isoforms. We conclude that the wupA associated HL results from the combined haploinsufficiency of a large set of TnI isoforms. The qualitative and quantitative normal expression of which, requires the chromosomal integrity of the wupA genomic region. Since all fly TnI isoforms are encoded in the same gene, its HL condition becomes unavoidable. These wupA features are comparable to those of dpp, the only other HL studied to some extent, and reveal a scenario of strict dosage dependence with implications for gene expression regulation and splitting.

Disrupting upstream translation in mRNAs is associated with human disease

Ribosome-profiling has uncovered pervasive translation in non-canonical open reading frames, however the biological significance of this phenomenon remains unclear. Using genetic variation from 71,702 human genomes, we assess patterns of selection in translated upstream open reading frames (uORFs) in 5’UTRs. We show that uORF variants introducing new stop codons, or strengthening existing stop codons, are under strong negative selection comparable to protein-coding missense variants. Using these variants, we map and validate gene-disease associations in two independent biobanks containing exome sequencing from 10,900 and 32,268 individuals, respectively, and elucidate their impact on protein expression in human cells. Our results suggest translation disrupting mechanisms relating uORF variation to reduced protein expression, and demonstrate that translation at uORFs is genetically constrained in 50% of human genes.

Use of signals of positive and negative selection to distinguish cancer genes and passenger genes

A major goal of cancer genomics is to identify all genes that play critical roles in carcinogenesis. Most approaches focused on genes positively selected for mutations that drive carcinogenesis and neglected the role of negative selection. Some studies have actually concluded that negative selection has no role in cancer evolution. We have re-examined the role of negative selection in tumor evolution through the analysis of the patterns of somatic mutations affecting the coding sequences of human genes. Our analyses have confirmed that tumor suppressor genes are positively selected for inactivating mutations, oncogenes, however, were found to display signals of both negative selection for inactivating mutations and positive selection for activating mutations. Significantly, we have identified numerous human genes that show signs of strong negative selection during tumor evolution, suggesting that their functional integrity is essential for the growth and survival of tumor cells.

Different selection dynamics of S and RdRp between SARS-CoV-2 genomes with and without the dominant mutations

SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic that has affected millions of people worldwide, with no dedicated treatment or vaccine currently available. As pharmaceutical research against and the most frequently used tests for SARS-CoV-2 infection both depend on the genomic and peptide sequences of the virus for their efficacy, understanding the mutation rates and content of the virus is critical. Two key proteins for SARS-CoV-2 infection and replication are the S protein, responsible for viral entry into the cells, and RdRp, the RNA polymerase responsible for replicating the viral genome. Due to their roles in the viral cycle, these proteins are crucial for the fitness and infectiousness of the virus. Our previous findings had shown that the two most frequently observed mutations in the SARS-CoV-2 genome, 14408C>T in the RdRp coding region, and 23403A>G in the S gene, are correlated with higher mutation density over time. In this study, we further detail the selection dynamics and the mutation rates of SARS-CoV-2 genes, comparing them between isolates carrying both mutations, and isolates carrying neither. We find that the S gene and the RdRp coding region show the highest variance between the genotypes, and their selection dynamics contrast each other over time. The S gene displays higher positive selection in mutant isolates early on, and undergoes increasing negative selection over time, whereas the RdRp region in the mutant isolates shows strong negative selection throughout the pandemic.