Systems Biology Approach Identifies Prognostic Signatures of Poor Overall Survival and Guides the Prioritization of Novel BET-CHK1 Combination Therapy for Osteosarcoma

Osteosarcoma (OS) patients exhibit poor overall survival, partly due to copy number variations (CNVs) resulting in dysregulated gene expression and therapeutic resistance. To identify actionable prognostic signatures of poor overall survival, we employed a systems biology approach using public databases to integrate CNVs, gene expression, and survival outcomes in pediatric, adolescent, and young adult OS patients. Chromosome 8 was a hotspot for poor prognostic signatures. The MYC-RAD21 copy number gain (8q24) correlated with increased gene expression and poor overall survival in 90% of the patients (n = 85). MYC and RAD21 play a role in replication-stress, which is a therapeutically actionable network. We prioritized replication-stress regulators, bromodomain and extra-terminal proteins (BETs), and CHK1, in order to test the hypothesis that the inhibition of BET + CHK1 in MYC-RAD21+ pediatric OS models would be efficacious and safe. We demonstrate that MYC-RAD21+ pediatric OS cell lines were sensitive to the inhibition of BET (BETi) and CHK1 (CHK1i) at clinically achievable concentrations. While the potentiation of CHK1i-mediated effects by BETi was BET-BRD4-dependent, MYC expression was BET-BRD4-independent. In MYC-RAD21+ pediatric OS xenografts, BETi + CHK1i significantly decreased tumor growth, increased survival, and was well tolerated. Therefore, targeting replication stress is a promising strategy to pursue as a therapeutic option for this devastating disease

The epigenetic regulator CXXC finger protein 1 is essential for murine hematopoiesis.

CXXC finger protein 1 (Cfp1), encoded by the Cxxc1 gene, binds to DNA sequences containing an unmethylated CpG dinucleotide and is an epigenetic regulator of both cytosine and histone methylation. Cxxc1-null mouse embryos fail to gastrulate, and Cxxc1-null embryonic stem cells are viable but cannot differentiate, suggesting that Cfp1 is required for chromatin remodeling associated with stem cell differentiation and embryogenesis. Mice homozygous for a conditional Cxxc1 deletion allele and carrying the inducible Mx1-Cre transgene were generated to assess Cfp1 function in adult animals. Induction of Cre expression in adult animals led to Cfp1 depletion in hematopoietic cells, a failure of hematopoiesis with a nearly complete loss of lineage-committed progenitors and mature cells, elevated levels of apoptosis, and death within two weeks. A similar pathology resulted following transplantation of conditional Cxxc1 bone marrow cells into wild type recipients, demonstrating this phenotype is intrinsic to Cfp1 function within bone marrow cells. Remarkably, the Lin- Sca-1+ c-Kit+ population of cells in the bone marrow, which is enriched for hematopoietic stem cells and multi-potential progenitor cells, persists and expands in the absence of Cfp1 during this time frame. Thus, Cfp1 is necessary for hematopoietic stem and multi-potential progenitor cell function and for the developmental potential of differentiating hematopoietic cells

CSK Controls Retinoic Acid Receptor (RAR) Signaling: a RAR-c-SRC Signaling Axis Is Required for Neuritogenic Differentiation▿

Herein, we report the first evidence that c-SRC is required for retinoic acid (RA) receptor (RAR) signaling, an observation that suggests a new paradigm for this family of nuclear hormone receptors. We observed that CSK negatively regulates RAR functions required for neuritogenic differentiation. CSK overexpression inhibited RA-mediated neurite outgrowth, a result which correlated with the inhibition of the SFK c-SRC. Consistent with an extranuclear effect of CSK on RAR signaling and neurite outgrowth, CSK overexpression blocked the downstream activation of RAC1. The conversion of GDP-RAC1 to GTP-RAC1 parallels the activation of c-SRC as early as 15 min following all-trans-retinoic acid treatment in LA-N-5 cells. The cytoplasmic colocalization of c-SRC and RARγ was confirmed by immunofluorescence staining and confocal microscopy. A direct and ligand-dependent binding of RAR with SRC was observed by surface plasmon resonance, and coimmunoprecipitation studies confirmed the in vivo binding of RARγ to c-SRC. Deletion of a proline-rich domain within RARγ abrogated this interaction in vivo. CSK blocked the RAR-RA-dependent activation of SRC and neurite outgrowth in LA-N-5 cells. The results suggest that transcriptional signaling events mediated by RA-RAR are necessary but not sufficient to mediate complex differentiation in neuronal cells. We have elucidated a nongenomic extranuclear signal mediated by the RAR-SRC interaction that is negatively regulated by CSK and is required for RA-induced neuronal differentiation

Ablation of the <i>Cxxc1</i> gene in adult mice causes death.

<p>Mice homozygous for the conditional <i>Cxxc1</i> allele and carrying or lacking the <i>Mx1-Cre</i> transgene were injected with poly(I:C) at days 0, 2, and 4. (A) Indicated tissues were collected at day 4 or day 7 following the initiation of Cre induction. Genomic DNA was isolated and PCR analysis was performed to assess the relative abundance of the conditional <i>Cxxc1</i> allele (<i>Cxxc1-flox</i>) and recombined <i>Cxxc1</i> (<i>ΔCxxc1</i>) allele, as well as the presence of the <i>Mx1-Cre</i> transgene (<i>Cre</i>). (B) Bone marrow cells were collected 48 hours after a single poly(I:C) injection and cultured ex vivo for 2 days. Cellular extracts were analyzed for Cfp1 protein levels by western analysis. Actin levels were determined as a loading control. (C) Animals were observed for survival following initiation of Cre induction. (N = 12 for each genotype) (D) Light microscopy of femur, spleen, and liver collected 11 days after initiation of Cre induction. There is a marked depletion of hematopoietic cells in the bone marrow and reduction of red pulp in the spleen of mutant mice. The liver appears normal.</p

Bone marrow exhibits decreased cellularity and increased apoptosis, but no change in global cytosine methylation, following ablation of the <i>Cxxc1</i> gene.

<p>(A) Following transplantation of control or mutant bone marrow (N = 2 and N = 3 recipients, respectively), poly(I:C) was injected on days 0, 2, and 4, and on day 9 following initiation of Cre induction total bone marrow cells were isolated and analyzed (P = 0.04). Similarly, control or mutant mice were induced with poly(I:C), and on day 8 following initiation of Cre induction, total bone marrow was analyzed; N = 11 controls and N = 16 mutant mice (P<0.0001). (B) Bone marrow cells were collected from mice of the indicated genotypes on day 8 after day 0, 2, and 4 poly(I:C) injections, and cells were analyzed for apoptosis by flow cytometry using Annexin V and PI staining (“in vivo BM”); N = 6 for Cre- and N = 5 for Cre+ for each of two independent experiments. Alternatively, after a poly(I:C) injection on day 0, bone marrow was collected on day 2 from mice of the indicated genotypes, cultured ex vivo, and then analyzed for apoptosis by Annexin V and PI staining at days 3, 4, and 5 following initiation of Cre induction (“in vitro cultured BM”); for each genotype, N = 4 for day 3 and N = 7 for days 4 and 5 (P≤0.0003) for each of two independent experiments. Asterisks denote statistically significant differences in cell number or frequency in total LDBMCs. (C) Genomic DNA was isolated from ex vivo cultured bone marrow cells as described in (B). Global genomic cytosine methylation levels were determined using a methyl-acceptance assay, as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113745#pone.0113745-Carlone1" target="_blank">[9]</a>. Similar analysis was done using DNA isolated from wild type or Cfp1-null ES cells as controls. Asterisk denotes a statistically significant difference (P<0.001).</p

Hematopoietic cells enriched for stem cells are tolerant of Cfp1 depletion.

<p>Mutant and control mice were induced with poly(I:C) injections at days 0, 2, and 4. Bone marrow cells were collected at day 8 following initiation of Cre induction and analyzed by flow cytometry for the indicated cell surface markers. (A) Scatter plot showing a representative experiment. (B–C) Summary for enumeration of LSK (Lin- Sca1+ Kit+), LT-HSC (Lin- Sca1+ Kit+ CD34- Flt3-), ST-HSC (Lin- Sca1+ Kit+ CD34+ Flt3+), and more primitive ST-HSC (Lin- Sca1+ Kit+ CD34+ Flt3-). (A) and (B) present the data in terms of percent of total LDBMCs, while (C) presents cell number per femur. (For B–C, N = 11 for Cre- controls and N = 16 for Cre+ mutants for each of three independent experiments.) (D) Total RNA was isolated from purified LSK cells derived from the bone marrow of mice of the indicated genotypes (day 8 after poly(I:C) injections on days 0, 2, and 4), and semi-quantitative RT-PCR was performed to determine the level of <i>Cxxc1</i> transcript. RNA from wild type or <i>Cxxc1-null</i> ES cells was used as positive and negative controls, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113745#pone.0113745-Carlone1" target="_blank">[9]</a>. The conditions for all PCR reactions were confirmed to be within the linear range (data not shown). (E) Mutant and control mice were induced with poly(I:C) injections on days 0, 2, and 4, and BrdU was injected on day 7. On day 8 following initiation of Cre induction, bone marrow was isolated and analyzed for the frequency of BrdU incorporation into LSK cells and CD34/Flt3 subpopulations. Asterisks denote statistically significant differences (P≤0.02). (F) LSK cells were recovered from the bone marrow of mutant or control mice 8 days following Cre induction and examined for apoptosis by Annexin V and PI staining.</p

Hematopoietic progenitor cell populations are sensitive to Cfp1 depletion.

<p>Mutant and control mice were induced with poly(I:C) injections at days 0, 2, and 4. Tissues were collected on day 8 following initiation of Cre induction for all analyses. (A) Bone marrow and spleen cells were each collected and analyzed by colony forming assay for myeloid hematopoietic progenitors. For both tissues, N = 3 for Cre- controls and N = 8 for Cre+ mutants. (B–D) Bone marrow cells were collected, stained for the indicated cell surface markers, and analyzed by flow cytometry. (B) Scatter plot showing a representative experiment. (C) Summary for frequency per total LDBMCs (percent) of CMP (Lin- Sca1- Kit+ IL7Ra- FcgRII/III lo CD34+), GMP (Lin- Sca1- Kit+ IL7Ra- FcgRII/III hi CD34+), MEP (Lin- Sca1- Kit+ IL7Ra- FcgRII/III lo CD34-), and CLP (Lin- Sca1 lo Kit lo IL7Ra+). (D) Summary for number of stained cells per femur. (For C–D, N = 11 for Cre- controls and N = 16 for Cre+ mutants for three independent experiments.) Asterisks denote statistically significant differences in cell number or frequency (P≤0.03).</p

The accumulation of LSK CD150+ cells following <i>Cxxc1</i> deletion is distinct from transient interferon effects.

<p>Mutant and control mice were induced with poly(I:C) injections on days 0, 2, and 4, and bone marrow was analyzed by flow cytometry for LSK-CD150+ frequency on days 4, 6, or 8 following initiation of Cre induction. N = 4 or 5 for each genotype and each time point. Asterisks denote statistically significant differences (P≤0.01).</p

Establishment and characterization of patient-derived xenograft of a rare pediatric anaplastic pleomorphic xanthoastrocytoma (PXA) bearing a CDC42SE2-BRAF fusion

Abstract Pleomorphic xanthoastrocytoma (PXA) is a rare subset of primary pediatric glioma with 70% 5-year disease free survival. However, up to 20% of cases present with local recurrence and malignant transformation into more aggressive type anaplastic PXA (AXPA) or glioblastoma. The understanding of disease etiology and mechanisms driving PXA and APXA are limited, and there is no standard of care. Therefore, development of relevant preclinical models to investigate molecular underpinnings of disease and to guide novel therapeutic approaches are of interest. Here, for the first time we established, and characterized a patient-derived xenograft (PDX) from a leptomeningeal spread of a patient with recurrent APXA bearing a novel CDC42SE2-BRAF fusion. An integrated -omics analysis was conducted to assess model fidelity of the genomic, transcriptomic, and proteomic/phosphoproteomic landscapes. A stable xenoline was derived directly from the patient recurrent tumor and maintained in 2D and 3D culture systems. Conserved histology features between the PDX and matched APXA specimen were maintained through serial passages. Whole exome sequencing (WES) demonstrated a high degree of conservation in the genomic landscape between PDX and matched human tumor, including small variants (Pearson’s r = 0.794–0.839) and tumor mutational burden (~ 3 mutations/MB). Large chromosomal variations including chromosomal gains and losses were preserved in PDX. Notably, chromosomal gain in chromosomes 4–9, 17 and 18 and loss in the short arm of chromosome 9 associated with homozygous 9p21.3 deletion involving CDKN2A/B locus were identified in both patient tumor and PDX sample. Moreover, chromosomal rearrangement involving 7q34 fusion; CDC42SE-BRAF t (5;7) (q31.1, q34) (5:130,721,239, 7:140,482,820) was identified in the PDX tumor, xenoline and matched human tumor. Transcriptomic profile of the patient’s tumor was retained in PDX (Pearson r = 0.88) and in xenoline (Pearson r = 0.63) as well as preservation of enriched signaling pathways (FDR Adjusted P < 0.05) including MAPK, EGFR and PI3K/AKT pathways. The multi-omics data of (WES, transcriptome, and reverse phase protein array (RPPA) was integrated to deduce potential actionable pathways for treatment (FDR < 0.05) including KEGG01521, KEGG05202, and KEGG05200. Both xenoline and PDX were resistant to the MEK inhibitors trametinib or mirdametinib at clinically relevant doses, recapitulating the patient’s resistance to such treatment in the clinic. This set of APXA models will serve as a preclinical resource for developing novel therapeutic regimens for rare anaplastic PXAs and pediatric high-grade gliomas bearing BRAF fusions