Leveraging big data of immune checkpoint blockade response identifies novel potential targets.

BACKGROUND: The development of immune checkpoint blockade (ICB) has changed the way we treat various cancers. While ICB produces durable survival benefits in a number of malignancies, a large proportion of treated patients do not derive clinical benefit. Recent clinical profiling studies have shed light on molecular features and mechanisms that modulate response to ICB. Nevertheless, none of these identified molecular features were investigated in large enough cohorts to be of clinical value. MATERIALS AND METHODS: Literature review was carried out to identify relevant studies including clinical dataset of patients treated with ICB [anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1), anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) or the combination] and available sequencing data. Tumor mutational burden (TMB) and 37 previously reported gene expression (GE) signatures were computed with respect to the original publication. Biomarker association with ICB response (IR) and survival (progression-free survival/overall survival) was investigated separately within each study and combined together for meta-analysis. RESULTS: We carried out a comparative meta-analysis of genomic and transcriptomic biomarkers of IRs in over 3600 patients across 12 tumor types and implemented an open-source web application (predictIO.ca) for exploration. TMB and 21/37 gene signatures were predictive of IRs across tumor types. We next developed a de novo GE signature (PredictIO) from our pan-cancer analysis and demonstrated its superior predictive value over other biomarkers. To identify novel targets, we computed the T-cell dysfunction score for each gene within PredictIO and their ability to predict dual PD-1/CTLA-4 blockade in mice. Two genes, F2RL1 (encoding protease-activated receptor-2) and RBFOX2 (encoding RNA-binding motif protein 9), were concurrently associated with worse ICB clinical outcomes, T-cell dysfunction in ICB-naive patients and resistance to dual PD-1/CTLA-4 blockade in preclinical models. CONCLUSION: Our study highlights the potential of large-scale meta-analyses in identifying novel biomarkers and potential therapeutic targets for cancer immunotherapy.

Leukemic HRX fusion proteins inhibit GADD34-induced apoptosis and associate with the GADD34 and hSNF5/INI1 proteins.

One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene (also called MLL, ALL-1, or HTRX) at chromosomal locus 11q23, resulting in the formation of HRX fusion proteins. Using the yeast two-hybrid system and human cell culture coimmunoprecipitation experiments, we show here that HRX proteins interact directly with the GADD34 protein. We have found that transfected cells overexpressing GADD34 display a significant increase in apoptosis after treatment with ionizing radiation, indicating that GADD34 expression not only correlates with apoptosis but also can enhance apoptosis. The amino-terminal third of the GADD34 protein was necessary for this observed increase in apoptosis. Furthermore, coexpression of three different HRX fusion proteins (HRX-ENL, HRX-AF9, and HRX-ELL) had an anti-apoptotic effect, abrogating GADD34-induced apoptosis. In contrast, expression of wild-type HRX gave rise to an increase in apoptosis. The difference observed here between wild-type HRX and the leukemic HRX fusion proteins suggests that inhibition of GADD34-mediated apoptosis may be important to leukemogenesis. We also show here that GADD34 binds the human SNF5/INI1 protein, a member of the SNF/SWI complex that can remodel chromatin and activate transcription. These studies demonstrate, for the first time, a gain of function for leukemic HRX fusion proteins compared to wild-type protein. We propose that the role of HRX fusion proteins as negative regulators of post-DNA-damage-induced apoptosis is important to leukemia progression.

HRX leukemic fusion proteins form a heterocomplex with the leukemia-associated protein SET and protein phosphatase 2A.

One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene at chromosome locus 11q23, resulting in HRX fusion proteins. Using the yeast two-hybrid system, in vitro binding studies, and human cell culture coimmunoprecipitation experiments, we show here that a region of the HRX protein that is consistently retained in HRX leukemic fusion proteins interacts directly with SET, another protein implicated in leukemia. We have identified the binding sites on HRX for SET and show that these sequences are clustered near the A.T hooks that have been shown to bind DNA. We also show that carboxyl-terminal SET sequences, possibly the acidic tail of SET, bind to HRX. We have also found serine/threonine-specific protein phosphatase activity in anti-HRX coimmunoprecipitates. Using the phosphatase inhibitor okadaic acid and Western blotting, the phosphatase was identified as protein phosphatase 2A (PP2A). Mutation of a single amino acid in one of the SET binding sites of HRX resulted in lower amounts of both coimmunoprecipitated SET protein and coimmunoprecipitated PP2A. These results suggest that the leukemogenic effects of HRX fusion proteins may be related to interactions with SET and PP2A.

HRX involvement in de novo and secondary leukemias with diverse chromosome 11q23 abnormalities.

Chromosome band 11q23 is a site of recurrent translocations and interstitial deletions in human leukemias. Recent studies have shown that the 11q23 gene HRX is fused to heterologous genes from chromosomes 4 or 19 after t(4;11)(q21;q23) and t(11;19)(q23;p13) translocations to create fusion genes encoding proteins with structural features of chimeric transcription factors. In this report, we show structural alterations of HRX by conventional Southern blot analyses in 26 of 27 de novo leukemias with cytogenetically diverse 11q23 abnormalities. The sole case that lacked HRX rearrangements was a t(11;17)-acute myeloid leukemia with French-American-British M3-like morphology. We also analyzed 10 secondary leukemias that arose after therapy with topoisomerase II inhibitors and found HRX rearrangements in 7 of 7 with 11q23 translocations, and in 2 of 2 with unsuccessful karyotypes. In total, we observed HRX rearrangements in 35 leukemias involving at least nine distinct donor loci (1q32, 4q21, 6q27, 7p15, 9p21-24, 15q15, 16p13, and two 19p13 sites). All breakpoints localized to an 8-kb region that encompassed exons 5-11 of HRX, suggesting that fusion proteins containing similar portions of HRX may be consistently created in leukemias with 11q23 abnormalities. We conclude that alteration of HRX is a recurrent pathogenetic event in leukemias with 11q23 aberrations involving many potential partners in a variety of settings including acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia in blast crisis, and topoisomerase II inhibitor-induced secondary leukemias of both the myeloid and lymphoid lineages.

A serine/proline-rich protein is fused to HRX in t(4;11) acute leukemias.

Translocations involving chromosome band 11q23 in acute leukemias have recently been shown to involve the HRX gene that codes for a protein with significant similarity to Drosophila trithorax. HRX gene alterations are consistently observed in t(4;11) (q21;q23)-carrying leukemias and cell lines by Southern blot analyses and are accompanied by HRX transcripts of anomalous size on Northern blots. HRX-homologous cDNAs were isolated from a library prepared from t(4;11)-carrying acute leukemia cells. cDNAs representative of transcription products from the derivative 11 chromosome were shown to contain HRX sequences fused to sequences derived from chromosome band 4q21. Fragments of the latter were used to clone and analyze cDNAs for wild-type 4q21 transcripts that predicted a 140-Kd basic protein (named FEL) that is rich in prolines, serines, and charged amino acids. FEL contains guanosine triphosphate-binding and nuclear localization consensus sequences and uses one of two possible 5' exons encoding the first 12 or 5 amino acids. After t(4;11) translocations, 913 C-terminal amino acids of FEL are fused in frame to the N-terminal portion of HRX containing its minor groove DNA binding motifs. These features are similar to predicted t(11;19) fusion proteins, suggesting that HRX consistently contributes a novel DNA-binding motif to at least two different chimeric proteins in acute leukemias.

Libraries for each human chromosome, constructed from sorter-enriched chromosomes by using linker-adaptor PCR.

We describe here the production of complex libraries enriched in sequences from each human chromosome type, starting with only a few thousand sorter-purified chromosomes. In this procedure, DNA is extracted from the sorted chromosomes, digested to completion by using the frequently cutting restriction endonuclease Sau3A1, and ligated, on each end, to an adaptor oligonucleotide. These fragments are then amplified using PCR with a sequence homologous to the adaptor oligonucleotide as a primer. We have used this procedure to produce PCR libraries for each of the 24 human chromosomes. These libraries were characterized by gel electrophoresis and found to be composed of a continuum of sequences ranging in size from a few hundred to approximately 1,000 bp. The libraries, when used as probes for fluorescence in situ hybridization, stained the target chromosomes more or less continuously, even after PCR amplification for more than 200 cycles. These libraries are useful as hybridization probes to facilitate molecular cytogenetic studies and as sources of probes either for identification of polymorphic short tandemly repeated sequences or for development of sequence-tagged sites.

Involvement of a homolog of Drosophila trithorax by 11q23 chromosomal translocations in acute leukemias.

We have identified a human homolog of the Drosophila trithorax protein that is structurally altered by 11q23 translocations in acute leukemias. Human trithorax (HRX) is a predicted 431 kd protein containing two potential DNA-binding motifs consisting of zinc fingers conserved with the fly protein and nonconserved amino-terminal "AT hook" motifs related to the DNA-binding motifs in HMG proteins. 11q23 translocations disrupt the HRX gene between these two motifs, and in a t(11;19)-carrying cell line fusion transcripts are expressed from both derivative chromosomes. The more abundant derivative 11 transcript codes for a chimeric protein containing the AT hook motifs fused to a previously undescribed protein (ENL) from chromosome 19. These data suggest a novel role for a trithorax-homologous protein in multilineage human leukemias that may be mediated by DNA binding within the minor groove at AT-rich sites, implicated to play an important role in bacterial IHF-, yeast datin-, and mammalian HMG-mediated gene activation.

Clinical applications of fluorescence in situ hybridization.

We review here the application of fluorescence in situ hybridization with chromosome-specific probes to chromosome classification and to detection of changes in chromosome number or structure associated with genetic disease. Information is presented on probe types that are available for disease detection. We discuss the application of these probes to detection of numerical aberrations important for prenatal diagnosis and to detection and characterization of numerical and structural aberrations in metaphase spreads and in interphase nuclei to facilitate tumor diagnosis.

Applications of fluorescence in situ hybridization in biological dosimetry and detection of disease-specific chromosome aberrations.

Dual color FISH with whole chromosome and pan-centromere probes facilitates rapid detection of stable structural aberrations such as translocations. This approach should allow analysis of translocations for assessment of genetic damage at long times after exposure or as a result of chronic exposure during a long period of time. Multi-color FISH with locus specific probes allows assessment of the frequency of cells carrying specific aberrations known to be associated with tumorigenesis, analysis of the series of genetic changes that occur during tumor evolution and correlation between genotype and phenotype. The power of FISH for analysis of random and tumor related events will increase steadily as informative probes are developed during the course of the International Human Genome Project.

Detection of bcr-abl fusion in chronic myelogeneous leukemia by in situ hybridization.

Chronic myelogeneous leukemia (CML) is genetically characterized by fusion of the bcr and abl genes on chromosomes 22 and 9, respectively. In most cases, the fusion involves a reciprocal translocation t(9;22)(q34;q11), which produces the cytogenetically distinctive Philadelphia chromosome (Ph1). Fusion can be detected by Southern (DNA) analysis or by in vitro amplification of the messenger RNA from the fusion gene with polymerase chain reaction (PCR). These techniques are sensitive but cannot be applied to single cells. Two-color fluorescence in situ hybridization (FISH) was used with probes from portions of the bcr and abl genes to detect the bcr-abl fusion in individual blood and bone marrow cells from six patients. The fusion event was detected in all samples analyzed, of which three were cytogenetically Ph1-negative. One of the Ph1-negative samples was also PCR-negative. This approach is fast and sensitive, and provides potential for determining the frequency of the abnormality in different cell lineages.

Analytical approaches to detection and characterization of disease-linked chromosome aberrations.

Flow karyotyping and FISH with chromosome specific or disease-locus-specific probes are powerful adjuncts to conventional cytogenetic analysis. Flow karyotyping is well suited to quantitative analysis of DNA content changes that occur during structural rearrangement. FISH with probes for repeated sequences allows ready detection of aneuploidy in interphase cells. FISH with whole chromosome composite probes to metaphase spreads facilitates detection of subtle structural changes and allows detection of structural aberrations that occur at frequencies as low as 10(-3). FISH with locus-specific probes facilitates diagnosis of specific genetic diseases, allows phenotype-genotype correlation on a cell-by-cell basis and may be developed into a sensitive method for detection of residual disease.

Sequence and organization of the diversity, joining, and constant region genes of the human T-cell delta-chain locus.

In this paper we describe the genomic organization and sequence of the human T-cell receptor delta-chain diversity, joining, and constant genes. There is one delta-chain constant region gene (C delta) located approximately equal to 85 kilobases (kb) upstream of the alpha-chain constant region. The delta-chain constant region consists of four exons, whose organization is very similar to that of the C alpha exons, suggesting that C alpha and C delta may have arisen from a gene duplication event. The first exon encodes most of the extracellular constant domain, the second encodes a hinge-like region, and the third encodes the entire transmembrane segment and intracytoplasmic portion, whereas the last exon contains exclusively 3' untranslated sequences. Three joining segments, J delta 1, J delta 2, and J delta 3, are found approximately equal to 12, approximately equal to 5.7, and approximately equal to 3.4 kb upstream of the first exon of C delta. Two functional diversity gene segments, D delta 1 and D delta 2, which can be productively translated in all three reading frames, are found 1 and 9.6 kb upstream of J delta 1. The presence of two D delta with such potential for diversity may offset the limited repertoire of the J delta and V delta genes. The spacer distribution in the recombinational signals flanking D delta and J delta segments allows recombination with V alpha gene segments; however, examination of delta-chain messages does not indicate that this is the case, suggesting that the delta chain uses unique variable gene segments and raising the question as to the reasons for this phenomenon.

Rearrangement of T-cell delta locus in lymphoproliferative disorders.

Studies of lymphoproliferative disorders using immunoglobulin and T-cell receptor genes have contributed to our understanding of clonality and lineages of these disorders. In this study, we examined the rearrangement of the recently discovered T-cell delta chain genes in a variety of lymphoproliferative diseases. We show here that six of 14 T-cell lymphomas and five of 23 B-cell lymphomas or B-cell leukemia cell lines have rearranged the delta loci, while two of two hyperimmune reactions retain germline configuration within these genes. Seven of ten cases of AILD were rearranged, and Lennert's lymphoma, which has been previously described as a T-cell malignancy, also contains rearrangements in the delta chain genes (three of five). Large cell anaplastic lymphomas positive for the activation antigen CD 30 also contain rearrangement in about one-half (five of 11) of the tumors examined. Two of seven of the Hodgkin's lymphomas studied contained a rearrangement for this gene. This study indicates that this newly identified T-cell delta gene is useful in evaluating clonality but is not lineage specific. However, with only one exception (in 28 rearrangements), this gene rearranges in tumors with gamma and beta chain gene rearrangements, indicating that when used in conjunction with the other TcR genes, delta rearrangement may also be useful in evaluating lineages.

The human T cell receptor alpha-delta locus: a physical map of the variable, joining and constant region genes.

In this study a physical macro-restriction map of the entire human alpha locus that spans about 1000 kilobase pairs and includes the V alpha, J alpha and C alpha genes is presented. Evidence is provided that gene duplications were involved in the increase of genomic diversity of V alpha genes. In addition, we show a detailed map of a 40-kb region located approximately 100 kg upstream of the human C alpha gene. Direct evidence is provided to support that the human alpha chain locus, like the murine, also contains another T cell constant region gene in the alpha chain locus, the human delta chain gene. In addition, two J segments and one D segment have been identified. Using these genomic probes, we show that several T cell lines, including those known to express the surface gamma/delta heterodimer, have rearranged this region. The design of two separate centers of rearrangement within one locus that are involved in rearrangement events at different times, and the presence of high number of J segments in this region, may render the locus highly vulnerable to chromosomal translocation during T cell development.

Sequence and organization of the human T cell delta chain gene.

A novel human T cell receptor (TcR) gene, located 85 kbp upstream to the C alpha coding regions, was isolated using human genomic clones to identify cDNA homologous to messages encoded by this region. The deduced protein sequence of this gene is highly homologous to that of the newly identified constant region found in the murine TcR alpha chain locus. This gene undergoes rearrangements and is expressed at the RNA level in human thymocytes, peripheral T cells and several leukemic T cell lines which have been shown to express the surface gamma-delta heterodimer, suggesting that this gene encodes the human T cell delta chain.

Simple repeated sequences in human satellite DNA.

In an extensive analysis, using a range of restriction endonucleases, HinfI and TaqI were found to differentiate satellites I, II and III & IV. Satellite I is resistant to digestion by TaqI, but is cleaved by HinfI to yield three major fragments of approximate size 770, 850 and 950bp, associated in a single length of DNA. The 770bp fragment contains recognition sites for a number of other enzymes, whereas the 850 and 950bp fragments are "silent" by restriction enzyme analysis. Satellite II is digested by HinfI into a large number of very small (10-80bp) fragments, many of which also contain TaqI sites. A proportion of the HinfI sites in satellite II have the sequence 5'GA(GC)TC. The HinfI digestion products of satellites III and IV form a complete ladder, stretching from 15bp or less to more than 250bp, with adjacent multimers separated by an increment of 5bp. The ladder fragments do not contain TaqI sites and all HinfI sites have the sequence 5'GA(AT)TC. Three fragments from the HinfI ladder of satellite III have been sequenced, and all consist of a tandemly repeated 5bp sequence, 5'TTCCA, with a non-repeated, G+C rich sequence, 9bp in length, at the 3' end.