SETD2 mutations do not contribute to clonal fitness in response to chemotherapy in childhood B cell acute lymphoblastic leukemia.

Mutations in genes encoding epigenetic regulators are commonly observed at relapse in B cell acute lymphoblastic leukemia (B-ALL). Loss-of-function mutations in SETD2, an H3K36 methyltransferase, have been observed in B-ALL and other cancers. Previous studies on mutated SETD2 in solid tumors and acute myelogenous leukemia support a role in promoting resistance to DNA damaging agents. We did not observe chemoresistance, an impaired DNA damage response, nor increased mutation frequency in response to thiopurines using CRISPR-mediated knockout in wild-type B-ALL cell lines. Likewise, restoration of SETD2 in cell lines with hemizygous mutations did not increase sensitivity. SETD2 mutations affected the chromatin landscape and transcriptional output that was unique to each cell line. Collectively our data does not support a role for SETD2 mutations in driving clonal evolution and relapse in B-ALL, which is consistent with the lack of enrichment of SETD2 mutations at relapse in most studies.

Palbociclib in combination with chemotherapy in pediatric and young adult patients with relapsed/refractory acute lymphoblastic leukemia and lymphoma: A Children's Oncology Group study (AINV18P1).

BACKGROUND: Cyclin D has been shown to play an essential role in acute lymphoblastic leukemia (ALL) initiation and progression, providing rationale for targeting the CDK4/6-cyclin D complex that regulates cell cycle progression. PROCEDURE: The Children's Oncology Group AINV18P1 phase 1 trial evaluated the CDK4/6 inhibitor, palbociclib, in combination with standard four-drug re-induction chemotherapy in children and young adults with relapsed/refractory B- and T-cell lymphoblastic leukemia (ALL) and lymphoma. Palbociclib (50 mg/m2 /dose) was administered orally once daily for 21 consecutive days, first as a single agent (Days 1-3) and subsequently combined with re-induction chemotherapy. This two-part study was designed to determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D), followed by an expansion pharmacokinetic cohort. RESULTS: Twelve heavily pretreated patients enrolled, all of whom were evaluable for toxicity. One dose-limiting hematologic toxicity (DLT) occurred at the starting dose of 50 mg/m2 /dose orally for 21 days. No additional DLTs were observed in the dose determination or pharmacokinetic expansion cohorts, and overall rates of grade 3/4 nonhematologic toxicities were comparable to those observed with the chemotherapy platform alone. Five complete responses were observed, two among four patients with T-ALL and three among seven patients with B-ALL. Pharmacokinetic studies showed similar profiles with both liquid and capsule formulations of palbociclib. CONCLUSIONS: Palbociclib in combination with re-induction chemotherapy was well tolerated with a RP2D of 50 mg/m2 /day for 21 days. Complete responses were observed among heavily pretreated patients.

NSD2 E1099K drives relapse in pediatric acute lymphoblastic leukemia by disrupting 3D chromatin organization.

BACKGROUND: The NSD2 p.E1099K (EK) mutation is shown to be enriched in patients with relapsed acute lymphoblastic leukemia (ALL), indicating a role in clonal evolution and drug resistance. RESULTS: To uncover 3D chromatin architecture-related mechanisms underlying drug resistance, we perform Hi-C on three B-ALL cell lines heterozygous for NSD2 EK. The NSD2 mutation leads to widespread remodeling of the 3D genome, most dramatically in terms of compartment changes with a strong bias towards A compartment shifts. Systematic integration of the Hi-C data with previously published ATAC-seq, RNA-seq, and ChIP-seq data show an expansion in H3K36me2 and a shrinkage in H3K27me3 within A compartments as well as increased gene expression and chromatin accessibility. These results suggest that NSD2 EK plays a prominent role in chromatin decompaction through enrichment of H3K36me2. In contrast, we identify few changes in intra-topologically associating domain activity. While compartment changes vary across cell lines, a common core of decompacting loci are shared, driving the expression of genes/pathways previously implicated in drug resistance. We further perform RNA sequencing on a cohort of matched diagnosis/relapse ALL patients harboring the relapse-specific NSD2 EK mutation. Changes in patient gene expression upon relapse significantly correlate with core compartment changes, further implicating the role of NSD2 EK in genome decompaction. CONCLUSIONS: In spite of cell-context-dependent changes mediated by EK, there appears to be a shared transcriptional program dependent on compartment shifts which could explain phenotypic differences across EK cell lines. This core program is an attractive target for therapeutic intervention.

Activation of the mitogen-activated protein kinase-extracellular signal-regulated kinase pathway in childhood B-cell acute lymphoblastic leukemia.

RAS mutations are frequently observed in childhood B-cell acute lymphoblastic leukemia (B-ALL) and previous studies have yielded conflicting results as to whether they are associated with a poor outcome. We and others have demonstrated that the mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK) pathway can be activated through epigenetic mechanisms in the absence of RAS pathway mutations. Herein, we examined whether MAPK activation, as determined by measuring phosphorylated extracellular signal-regulated kinase (pERK) levels in 80 diagnostic patient samples using phosphoflow cytometry, could be used as a prognostic biomarker for pediatric B-ALL. The mean fluorescence intensity of pERK (MFI) was measured at baseline and after exogenous stimulation with or without pretreatment with the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib. Activation levels (MFI stimulated/MFI baseline) ranged from 0.76 to 4.40 (median = 1.26), and inhibition indexes (MFI stimulated/MFI trametinib stimulated) ranged from 0.439 to 5.640 (median = 1.30), with no significant difference between patients with wildtype versus mutant RAS for either. Logistic regression demonstrated that neither MAPK activation levels nor RAS mutation status at diagnosis alone or in combination was prognostic of outcome. However, 35% of RAS wildtype samples showed MAPK inhibition indexes greater than the median, thus raising the possibility that therapeutic strategies to inhibit MAPK activation may not be restricted to patients whose blasts display Ras pathway defects.

Minimal Residual Disease in Acute Lymphoblastic Leukemia: Current Practice and Future Directions.

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and advances in its clinical and laboratory biology have grown exponentially over the last few decades. Treatment outcome has improved steadily with over 90% of patients surviving 5 years from initial diagnosis. This success can be attributed in part to the development of a risk stratification approach to identify those subsets of patients with an outstanding outcome that might qualify for a reduction in therapy associated with fewer short and long term side effects. Likewise, recognition of patients with an inferior prognosis allows for augmentation of therapy, which has been shown to improve outcome. Among the clinical and biological variables known to impact prognosis, the kinetics of the reduction in tumor burden during initial therapy has emerged as the most important prognostic variable. Specifically, various methods have been used to detect minimal residual disease (MRD) with flow cytometric and molecular detection of antigen receptor gene rearrangements being the most common. However, many questions remain as to the optimal timing of these assays, their sensitivity, integration with other variables and role in treatment allocation of various ALL subgroups. Importantly, the emergence of next generation sequencing assays is likely to broaden the use of these assays to track disease evolution. This review will discuss the biological basis for utilizing MRD in risk assessment, the technical approaches and limitations of MRD detection and its emerging applications.

Evolution of the Epigenetic Landscape in Childhood B Acute Lymphoblastic Leukemia and Its Role in Drug Resistance.

Although B-cell acute lymphoblastic leukemia (B-ALL) is the most common malignancy in children and while highly curable, it remains a leading cause of cancer-related mortality. The outgrowth of tumor subclones carrying mutations in genes responsible for resistance to therapy has led to a Darwinian model of clonal selection. Previous work has indicated that alterations in the epigenome might contribute to clonal selection, yet the extent to which the chromatin state is altered under the selective pressures of therapy is unknown. To address this, we performed chromatin immunoprecipitation, gene expression analysis, and enhanced reduced representation bisulfite sequencing on a cohort of paired diagnosis and relapse samples from individual patients who all but one relapsed within 36 months of initial diagnosis. The chromatin state at diagnosis varied widely among patients, while the majority of peaks remained stable between diagnosis and relapse. Yet a significant fraction was either lost or newly gained, with some patients showing few differences and others showing massive changes of the epigenetic state. Evolution of the epigenome was associated with pathways previously linked to therapy resistance as well as novel candidate pathways through alterations in pyrimidine biosynthesis and downregulation of polycomb repressive complex 2 targets. Three novel, relapse-specific superenhancers were shared by a majority of patients including one associated with S100A8, the top upregulated gene seen at relapse in childhood B-ALL. Overall, our results support a role of the epigenome in clonal evolution and uncover new candidate pathways associated with relapse. SIGNIFICANCE: This study suggests a major role for epigenetic mechanisms in driving clonal evolution in B-ALL and identifies novel pathways associated with drug resistance.

Feasibility of monitoring peripheral blood to detect emerging clones in children with acute lymphoblastic leukemia†.

Relapse-enriched somatic variants drive drug resistance in childhood acute lymphoblastic leukemia. We used digital droplet-based polymerase chain reaction to establish whether relapse-enriched mutations in emerging subclones could be detected in peripheral blood samples before frank relapse. Although limitations in sensitivity for some probes hindered detection of certain variants, we successfully detected variants in NT5C2 and PRPS1 at a fractional abundance of 0.005% to 0.3%, 41 to 116 days before relapse. As mutations in both these genes confer resistance to thiopurines, early detection protocols using peripheral blood could be implemented to preemptively alter maintenance therapy to extinguish resistant clones before overt relapse.

Extensive Remodeling of the Immune Microenvironment in B Cell Acute Lymphoblastic Leukemia.

A subset of B cell acute lymphoblastic leukemia (B-ALL) patients will relapse and succumb to therapy-resistant disease. The bone marrow microenvironment may support B-ALL progression and treatment evasion. Utilizing single-cell approaches, we demonstrate B-ALL bone marrow immune microenvironment remodeling upon disease initiation and subsequent re-emergence during conventional chemotherapy. We uncover a role for non-classical monocytes in B-ALL survival, and demonstrate monocyte abundance at B-ALL diagnosis is predictive of pediatric and adult B-ALL patient survival. We show that human B-ALL blasts alter a vascularized microenvironment promoting monocytic differentiation, while depleting leukemia-associated monocytes in B-ALL animal models prolongs disease remission in vivo. Our profiling of the B-ALL immune microenvironment identifies extrinsic regulators of B-ALL survival supporting new immune-based therapeutic approaches for high-risk B-ALL treatment.

The NSD2 p.E1099K Mutation Is Enriched at Relapse and Confers Drug Resistance in a Cell Context-Dependent Manner in Pediatric Acute Lymphoblastic Leukemia.

The NSD2 p.E1099K (EK) mutation is observed in 10% of acute lymphoblastic leukemia (ALL) samples with enrichment at relapse indicating a role in clonal evolution and drug resistance. To discover mechanisms that mediate clonal expansion, we engineered B-precursor ALL (B-ALL) cell lines (Reh, 697) to overexpress wildtype (WT) and EK NSD2, but observed no differences in proliferation, clonal growth, or chemosensitivity. To address whether NSD2 EK acts collaboratively with other pathways, we used short hairpin RNAs to knockdown expression of NSD2 in B-ALL cell lines heterozygous for NSD2 EK (RS4;11, RCH-ACV, SEM). Knockdown resulted in decreased proliferation in all lines, decreased clonal growth in RCH-ACV, and increased sensitivity to cytotoxic chemotherapeutic agents, although the pattern of drug sensitivity varied among cell lines implying that the oncogenic properties of NSD2 mutations are likely cell context specific and rely on cooperative pathways. Knockdown of both Type II and REIIBP EK isoforms had a greater impact than knockdown of Type II alone, suggesting that both SET containing EK isoforms contribute to phenotypic changes driving relapse. Furthermore, in vivo models using both cell lines and patient samples revealed dramatically enhanced proliferation of NSD2 EK compared with WT and reduced sensitivity to 6-mercaptopurine in the relapse sample relative to diagnosis. Finally, EK-mediated changes in chromatin state and transcriptional output differed dramatically among cell lines further supporting a cell context-specific role of NSD2 EK. These results demonstrate a unique role of NSD2 EK in mediating clonal fitness through pleiotropic mechanisms dependent on the genetic and epigenetic landscape. IMPLICATIONS: NSD2 EK mutation leads to drug resistance and a clonal advantage in childhood B-ALL.

MSH6 haploinsufficiency at relapse contributes to the development of thiopurine resistance in pediatric B-lymphoblastic leukemia.

Survival of children with relapsed acute lymphoblastic leukemia is poor, and understanding mechanisms underlying resistance is essential to developing new therapy. Relapse-specific heterozygous deletions in MSH6, a crucial part of DNA mismatch repair, are frequently detected. Our aim was to determine whether MSH6 deletion results in a hypermutator phenotype associated with generation of secondary mutations involved in drug resistance, or if it leads to a failure to initiate apoptosis directly in response to chemotherapeutic agents. We knocked down MSH6 in mismatch repair proficient cell lines (697 and UOCB1) and showed significant increases in IC50s to 6-thioguanine and 6-mercaptopurine (697: 26- and 9-fold; UOCB1: 5- and 8-fold) in vitro, as well as increased resistance to 6-mercaptopurine treatment in vivo No shift in IC50 was observed in deficient cells (Reh and RS4;11). 697 MSH6 knockdown resulted in increased DNA thioguanine nucleotide levels compared to non-targeted cells (3070 vs 1722 fmol/µg DNA) with no difference observed in mismatch repair deficient cells. Loss of MSH6 did not give rise to microsatellite instability in cell lines or clinical samples, nor did it significantly increase mutation rate, but rather resulted in a defect in cell cycle arrest upon thiopurine exposure. MSH6 knockdown cells showed minimal activation of checkpoint regulator CHK1, γH2AX (DNA damage marker) and p53 levels upon treatment with thiopurines, consistent with intrinsic chemoresistance due to failure to recognize thioguanine nucleotide mismatching and initiate mismatch repair. Aberrant MSH6 adds to the list of alterations/mutations associated with acquired resistance to purine analogs emphasizing the importance of thiopurine therapy.

Three-Dimensional Assay for Studying Breast Cancer Cell Invasion.

Cancer cell invasion is a complex process that naturally occurs in a three-dimensional (3-D) environment comprised of tumor cells and extracellular matrix components (ECM). Therefore, examining the invasive ability of breast cancer cells in a 3-D assay is imperative to discovering novel treatment strategies aimed at preventing cancer invasion and metastasis. Here, I describe a method to quantitatively measure the number of invaded cancer cells within a 3-D microenvironment and determine the effects of potential drugs on this cellular process.

Genomic Characterization of Poorly Differentiated Neuroendocrine Carcinoma in a Pediatric Patient.

Primary neuroendocrine carcinomas (NEC) are rare tumors in children and young adults, resulting in a lack of standardized treatment approach. To refine the molecular taxonomy of these rare tumors, we performed whole exome sequencing in a pediatric patient with mediastinal NEC. We identified a somatic mutation in HRAS gene and LOH regions in NF2, MYO18B, and RUX3 genes. In addition, a germline heterozygous somatic variant in BRCA2 with LOH at that same position in the tumor tissue was also found. Our data provide valuable insight into the genomic landscape of this tumor, prompting further investigation of therapeutic targets.

MAPK signaling cascades mediate distinct glucocorticoid resistance mechanisms in pediatric leukemia.

The outcome for pediatric acute lymphoblastic leukemia (ALL) patients who relapse is dismal. A hallmark of relapsed disease is acquired resistance to multiple chemotherapeutic agents, particularly glucocorticoids. In this study, we performed a genome-scale short hairpin RNA screen to identify mediators of prednisolone sensitivity in ALL cell lines. The incorporation of these data with an integrated analysis of relapse-specific genetic and epigenetic changes allowed us to identify the mitogen-activated protein kinase (MAPK) pathway as a mediator of prednisolone resistance in pediatric ALL. We show that knockdown of the specific MAPK pathway members MEK2 and MEK4 increased sensitivity to prednisolone through distinct mechanisms. MEK4 knockdown increased sensitivity specifically to prednisolone by increasing the levels of the glucocorticoid receptor. MEK2 knockdown increased sensitivity to all chemotherapy agents tested by increasing the levels of p53. Furthermore, we demonstrate that inhibition of MEK1/2 with trametinib increased sensitivity of ALL cells and primary samples to chemotherapy in vitro and in vivo. To confirm a role for MAPK signaling in patients with relapsed ALL, we measured the activation of the MEK1/2 target ERK in matched diagnosis-relapse primary samples and observed increased phosphorylated ERK levels at relapse. Furthermore, relapse samples have an enhanced response to MEK inhibition compared to matched diagnosis samples in xenograft models. Together, our data indicate that inhibition of the MAPK pathway increases chemosensitivity to glucocorticoids and possibly other agents and that the MAPK pathway is an attractive target for prevention and/or treatment of relapsed disease.

Hypoxia promotes colon cancer dissemination through up-regulation of cell migration-inducing protein (CEMIP).

Hypoxic stress drives cancer progression by causing a transcriptional reprogramming. Recently, KIAA1199 was discovered to be a cell-migration inducing protein (renamed CEMIP) that is upregulated in human cancers. However, the mechanism of induction of CEMIP in cancer was hitherto unknown. Here we demonstrate that hypoxia induces CEMIP expression leading to enhanced cell migration. Immunohistochemistry of human colon cancer tissues revealed that CEMIP is upregulated in cancer cells located at the invasive front or in the submucosa. CEMIP localization inversely correlated with E-cadherin expression, which is characteristic of the epithelial-to-mesenchymal transition. Mechanistically, hypoxia-inducible-factor-2α (HIF-2α), but not HIF-1α binds directly to the hypoxia response element within the CEMIP promoter region resulting in increased CEMIP expression. Functional characterization reveals that CEMIP is a downstream effector of HIF-2α-mediated cell migration. Expression of CEMIP was demonstrated to negatively correlate with the expression of Jarid1A, a histone demethylase that removes methyl groups from H3K4me3 (an activation marker for transcription), resulting in altered gene repression. Low oxygen tension inhibits the function of Jarid1A, leading to increased presence of H3K4me3 within the CEMIP promoter. These results provide insight into the upregulation of CEMIP within cancer and can lead to novel treatment strategies targeting this cancer cell migration-promoting gene.

Development of a high-throughput three-dimensional invasion assay for anti-cancer drug discovery.

The lack of three-dimensional (3-D) high-throughput (HT) screening assays designed to identify anti-cancer invasion drugs is a major hurdle in reducing cancer-related mortality, with the key challenge being assay standardization. Presented is the development of a novel 3-D invasion assay with HT potential that involves surrounding cell-collagen spheres within collagen to create a 3-D environment through which cells can invade. Standardization was achieved by designing a tooled 96-well plate to create a precisely designated location for the cell-collagen spheres and by using dialdehyde dextran to inhibit collagen contraction, maintaining uniform size and shape. This permits automated readout for determination of the effect of inhibitory compounds on cancer cell invasion. Sensitivity was demonstrated by the ability to distinguish varying levels of invasiveness of cancer cell lines, and robustness was determined by calculating the Z-factor. A Z-factor of 0.65 was obtained by comparing the effects of DMSO and anti-ß1-integrin antibody, an inhibitory reagent, on the invasion of Du145 cancer cells, suggesting this novel assay is suitable for large scale drug discovery. As proof of principle, the NCI Diversity Compound Library was screened against human invasive cancer cells. Nine compounds exhibiting high potency and low toxicity were identified, including DX-52-1, a compound previously reported to inhibit cell migration, a critical determinant of cancer invasion. The results indicate that this innovative HT platform is a simple, precise, and easy to replicate 3-D invasion assay for anti-cancer drug discovery.

Unraveling the role of KIAA1199, a novel endoplasmic reticulum protein, in cancer cell migration.

BACKGROUND: Cell migration is a critical determinant of cancer metastasis, and a better understanding of the genes involved will lead to the identification of novel targets aimed at preventing cancer dissemination. KIAA1199 has been shown to be upregulated in human cancers, yet its role in cancer progression was hitherto unknown. METHODS: Clinical relevance was assessed by examining KIAA1199 expression in human cancer specimens. In vitro and in vivo studies were employed to determine the function of KIAA1199 in cancer progression. Cellular localization of KIAA1199 was microscopically determined. SNAP-tag pull-down assays were used to identify binding partner(s) of KIAA1199. Calcium levels were evaluated using spectrofluorometric and fluorescence resonance energy transfer analyses. Signaling pathways were dissected by Western blotting. Student t test was used to assess differences. All statistical tests were two-sided. RESULTS: KIAA1199 was upregulated in invasive breast cancer specimens and inversely associated with patient survival rate. Silencing of KIAA1199 in MDA-MB-435 cancer cells resulted in a mesenchymal-to-epithelial transition that reduced cell migratory ability in vitro (75% reduction; P < .001) and decreased metastasis in vivo (80% reduction; P < .001). Gain-of-function assays further demonstrated the role of KIAA1199 in cell migration. KIAA1199-enhanced cell migration required endoplasmic reticulum (ER) localization, where it forms a stable complex with the chaperone binding immunoglobulin protein (BiP). A novel ER-retention motif within KIAA1199 that is required for its ER localization, BiP interaction, and enhanced cell migration was identified. Mechanistically, KIAA1199 was found to mediate ER calcium leakage, and the resultant increase in cytosolic calcium ultimately led to protein kinase C alpha activation and cell migration. CONCLUSIONS: KIAA1199 serves as a novel cell migration-promoting gene and plays a critical role in maintaining cancer mesenchymal status.

Transcriptional and epigenetic regulation of KIAA1199 gene expression in human breast cancer.

Emerging evidence has demonstrated that upregulated expression of KIAA1199 in human cancer bodes for poor survival. The regulatory mechanism controlling KIAA1199 expression in cancer remains to be characterized. In the present study, we have isolated and characterized the human KIAA1199 promoter in terms of regulation of KIAA1199 gene expression. A 3.3 kb fragment of human genomic DNA containing the 5'-flanking sequence of the KIAA1199 gene possesses both suppressive and activating elements. Employing a deletion mutagenesis approach, a 1.4 kb proximal region was defined as the basic KIAA1199 promoter containing a TATA-box close to the transcription start site. A combination of 5'-primer extension study with 5'RACE DNA sequencing analysis revealed one major transcription start site that is utilized in the human KIAA1199 gene. Bioinformatics analysis suggested that the 1.4 kb KIAA1199 promoter contains putative activating regulatory elements, including activator protein-1(AP-1), Twist-1, and NF-κB sites. Sequential deletion and site-direct mutagenesis analysis demonstrated that the AP-1 and distal NF-κB sites are required for KIAA1199 gene expression. Further analyses using an electrophoretic mobility-shift assay and chromatin immunoprecipitation confirmed the requirement of these cis- and trans-acting elements in controlling KIAA1199 gene expression. Finally, we found that upregulated KIAA1199 expression in human breast cancer specimens correlated with hypomethylation of the regulatory region. Involvement of DNA methylation in regulation of KIAA1199 expression was recapitulated in human breast cancer cell lines. Taken together, our study unraveled the regulatory mechanisms controlling KIAA1199 gene expression in human cancer.