[PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA].

INTRODUCTION: Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, constituting approximately 25% of childhood cancers. In recent decades, survival rates have improved dramatically, from approximately 10% in the 1960's to over 90% today. This tremendous achievement has been accomplished through collaborative randomized clinical trials, with progressive evolution of highly efficient, risk-adapted multi-agent chemotherapeutic regimens, effective central nervous system prophylactic strategies and improved supportive care. Recently, our understanding of the genetic basis of ALL has been greatly enhanced, and precise methods for treatment response assessment with serial measurements of minimal residual disease have been developed. Certain patient subgroups have genetically targetable lesions, such as Philadelphia-positive ALL, whose outcomes have been dramatically improved by combined tyrosine kinase inhibitors and chemotherapy, or specific patient subsets of "Philadelphia-like" ALL. Despite the great progress in curing childhood ALL, significant challenges still remain. Acute adverse effects of chemotherapy may be life-threatening, and long-term side effects often impair survivors' quality of life. Survival rates in patients with relapsed or refractory ALL remain poor. This led to the introduction of novel immune-based therapies into the treatment of relapsed/refractory B-ALL: blinatumomab, a CD19 bi-specific T-cell engager; inotuzumab- a CD22-immunotoxin, and CD19-CAR (chimeric antigen receptor) T cells. These modalities have demonstrated improved remission rates with reduced toxicity compared to chemotherapy. The role of immunotherapy in the treatment of newly-diagnosed and relapsed patients will be more precisely defined in the near future.

Di-genic inheritance of germline POLE and PMS2 pathogenic variants causes a unique condition associated with pediatric cancer predisposition.

Polymerase proofreading-associated polyposis (PPAP) and Lynch syndrome, caused by mutated POLE and mismatch repair (MMR) genes, respectively, are associated with adult-onset cancer. PPAP and MMR-deficient tumors are both hypermutated, and each has a unique mutational signature. We describe a 4.5-year-old boy with multiple café au lait spots who presented with metastatic Sonic Hedgehog-activated medulloblastoma, with partial response to intensive chemotherapy and immunotherapy. The tumor showed microsatellite stability, loss of PMS2 nuclear expression, and an exceptionally high tumor mutational burden of 276 Mut/Mb. Germline molecular analysis revealed an inherited heterozygous pathogenic POLE variant and a de novo heterozygous PMS2 pathogenic variant. The tumor featured the MMR, POLE, and POLE+MMR mutational signatures. This is the first description of a di-genic condition, which we named "POL-LYNCH syndrome," manifested by an aggressive ultra-mutant pediatric medulloblastoma with a unique genomic signature.

Targeting the centriolar replication factor STIL synergizes with DNA damaging agents for treatment of ovarian cancer.

Advanced ovarian cancer is an incurable disease. Thus, novel therapies are required. We wished to identify new therapeutic targets for ovarian cancer. ShRNA screen performed in 42 ovarian cancer cell lines identified the centriolar replication factor STIL as an essential gene for ovarian cancer cells. This was verified in-vivo in orthotopic human ovarian cancer mouse models. STIL depletion by administration of siRNA in neutral liposomes resulted in robust anti-tumor effect that was further enhanced in combination with cisplatin. Consistent with this finding, STIL depletion enhanced the extent of DNA double strand breaks caused by DNA damaging agents. This was associated with centrosomal depletion, ongoing genomic instability and enhanced formation of micronuclei. Interestingly, the ongoing DNA damage was not associated with reduced DNA repair. Indeed, we observed that depletion of STIL enhanced canonical homologous recombination repair and increased BRCA1 and RAD51 foci in response to DNA double strand breaks. Thus, inhibition of STIL significantly enhances the efficacy of DNA damaging chemotherapeutic drugs in treatment of ovarian cancer.

Triplication of a 21q22 region contributes to B cell transformation through HMGN1 overexpression and loss of histone H3 Lys27 trimethylation.

Down syndrome confers a 20-fold increased risk of B cell acute lymphoblastic leukemia (B-ALL), and polysomy 21 is the most frequent somatic aneuploidy among all B-ALLs. Yet the mechanistic links between chromosome 21 triplication and B-ALL remain undefined. Here we show that germline triplication of only 31 genes orthologous to human chromosome 21q22 confers mouse progenitor B cell self renewal in vitro, maturation defects in vivo and B-ALL with either the BCR-ABL fusion protein or CRLF2 with activated JAK2. Chromosome 21q22 triplication suppresses histone H3 Lys27 trimethylation (H3K27me3) in progenitor B cells and B-ALLs, and 'bivalent' genes with both H3K27me3 and H3K4me3 at their promoters in wild-type progenitor B cells are preferentially overexpressed in triplicated cells. Human B-ALLs with polysomy 21 are distinguished by their overexpression of genes marked with H3K27me3 in multiple cell types. Overexpression of HMGN1, a nucleosome remodeling protein encoded on chromosome 21q22 (refs. 3,4,5), suppresses H3K27me3 and promotes both B cell proliferation in vitro and B-ALL in vivo.

The mitotic checkpoint gene, SIL is regulated by E2F1.

The SIL gene expression is increased in multiple cancers and correlates with the expression of mitotic spindle checkpoint genes and with increased metastatic potential. SIL regulates mitotic entry, organization of the mitotic spindle and cell survival. The E2F transcription factors regulate cell cycle progression by controlling the expression of genes mediating the G1/S transition. More recently, E2F has been shown to regulate mitotic spindle checkpoint genes as well. As SIL expression correlates with mitotic checkpoint genes, we hypothesized that SIL is regulated by E2F. We mined raw data of published experiments and performed new experiments by modification of E2F expression in cell lines, reporter assays and chromatin immunoprecipitation. Ectopic expression or endogenous activation of E2F induced the expression of SIL, while knockdown of E2F by shRNA, downregulated SIL expression. E2F activated SIL promoter by reporter assay and bound to SIL promoter in vivo. Taken together these data demonstrate that SIL is regulated by E2F. As SIL is essential for mitotic entry, E2F may regulate G2/M transition through the induction of SIL. Furthermore, as silencing of SIL cause apoptosis in cancer cells, these finding may have therapeutic relevance in tumors with constitutive activation of E2F.

Sil phosphorylation in a Pin1 binding domain affects the duration of the spindle checkpoint.

SIL is an immediate-early gene that is essential for embryonic development and is implicated in T-cell leukemia-associated translocations. We now show that the Sil protein is hyperphosphorylated during mitosis or in cells blocked at prometaphase by microtubule inhibitors. Cell cycle-dependent phosphorylation of Sil is required for its interaction with Pin1, a regulator of mitosis. Point mutation of the seven (S/T)P sites between amino acids 567 and 760 reduces mitotic phosphorylation of Sil, Pin1 binding, and spindle checkpoint duration. When a phosphorylation site mutant Sil is stably expressed, the duration of the spindle checkpoint is shortened in cells challenged with taxol or nocodazole, and the cells revert to a G2-like state. This event is associated with the downregulation of the kinase activity of the Cdc2/cyclin B1 complex and the dephosphorylation of the threonine 161 on the Cdc2 subunit. Sil downregulation by plasmid-mediated RNA interference limited the ability of cells to activate the spindle checkpoint and correlated with a reduction of Cdc2/cyclin B1 activity and phosphorylation on T161 on the Cdc2 subunit. These data suggest that a critical region of Sil is required to mediate the presentation of Cdc2 activity during spindle checkpoint arrest.

Histone deacetylase inhibitors--a new tool to treat cancer.

Transcriptional regulation in eukaryotes is a multilevel hierarchical process. It is becoming clear that higher-order chromatin structure, occurring via modifications of histones in their nucleosome structure, plays a crucial role in regulating gene expression, both in normal and pathological states. Deacetylation of histones by histone deacetylases (HDACs) modifies the chromatin from an open gene active euchromatin structure to a closed gene silenced heterochromatin structure. Several cancer promoting mutations and chromosomal translocations result in repression of transcription through abnormal recruitment and activation of HDACs, leading to neoplastic transformation. This is the rationale for the evolvement of HDAC inhibitors as a new class in cancer therapy. Trials have shown anti-proliferation effect of histone deacetylase inhibitors in cell culture, animal models and in human with both hematological and solid tumors. The exact mechanism by which histone deacetylase inhibitors exert their effect is still obscure. Reversal of the alteration in gene expression by fusion transcription factors or overexpressed repressors is just one of several possible explanations. The territory of heterochromatin in the vicinity of the nuclear periphery raised the possibility of involvement of nuclear envelope proteins in the regulation of transcription. Our laboratory is interested in the transcription repression mechanism induced by the nuclear envelope lamina associated polypeptide 2 (LAP2) family of proteins through chromatin modification. Here, we will describe the structure of the nucleosome, review regulation of gene expression by acetylation of histones and give an update on the current phase I and phase II clinical trials with histone deacetylase inhibitors.

Kidney, blood, and endothelium: developmental expression of stem cell leukemia during nephrogenesis.

BACKGROUND: In vertebrates the hematopoietic and renal tissues share a common mesodermal origin. Recently, we have analyzed global gene expression during human nephrogenesis and observed up-regulation of stem cell leukemia (SCL), a transcription factor critical for hematopoietic and endothelial lineage specification. Here we characterize the expression of SCL along with its distinct 3' hematopoietic and endothelial enhancer (SCL 3'En) during kidney development. METHODS: mRNA and protein expression of SCL were examined in developing murine and human kidneys by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. The activity of SCL 3'En was examined by X-galactosidase (X-gal) staining of embryonic kidneys obtained from SCL +6E5/lacZ/3'En transgenic mice and by reporter lacZ assay in various renal cell lines. RESULTS: We found developmental regulation of SCL mRNA with highest levels of expression in embryonic day 17 (E17) mouse kidneys and lowest in postnatal and adult kidneys. Immunostaining of human fetal kidneys demonstrated the protein predominantly in the nephrogenic cortex and particularly in mesenchymal cells and developing glomeruli. Similarly, SCL +6E5/lacZ/3'En transgenic kidneys showed prominent lacZ staining in cells resembling undifferentiated mesoderm cells in close proximity to S and comma-shaped primitive nephrons and in peritubular and glomerular vessel endothelium. The SCL 3'En was activated in the human embryonic kidney cell line (HEK 293), but not in cell lines derived from adult kidney. CONCLUSION: These observations suggest a possible role for SCL in renal vasculogenesis. Undifferentiated mesenchymal cells expressing SCL during early nephrogenesis might represent putative progenitors that can simultaneously give rise to kidney, blood, and endothelium.

Determination of the "time of useful consciousness" (TUC) in repeated exposures to simulated altitude of 25,000 ft (7,620 m).

Time of useful consciousness (TUC) was determined in 17 subjects exposed twice to 25,000 ft (7,620 m) in an altitude chamber. The criterion for TUC determination was inability to add two-digit numbers correctly. Median values of TUC were 267.5 seconds (s) in the first exposure and 240 s in the second. The intraindividual variability between the two exposures was 40.6 s. The probability of remaining in "useful" consciousness as a function of time at 25,000 ft (7,620 m) was similar in both exposures. The need for a more scientific approach towards the determination of time of useful consciousness in simulated high altitudes is raised.