KDM6A Regulates Immune Response Genes in Multiple Myeloma.

The histone H3K27 demethylase KDM6A is a tumor suppressor in multiple cancers, including multiple myeloma (MM). We created isogenic MM cells disrupted for KDM6A and tagged the endogenous protein to facilitate genome wide studies. KDM6A binds genes associated with immune recognition and cytokine signaling. Most importantly, KDM6A binds and activates NLRC5 and CIITA encoding regulators of Major Histocompatibility Complex (MHC) genes. Patient data indicate that NLRC5 and CIITA, are downregulated in MM with low KDM6A expression. Chromatin analysis shows that KDM6A binds poised and active enhancers and KDM6A loss led to decreased H3K27ac at enhancers, increased H3K27me3 levels in body of genes bound by KDM6A and decreased gene expression. Reestablishing histone acetylation with an HDAC3 inhibitor leads to upregulation of MHC expression, offering a strategy to restore immunogenicity of KDM6A deficient tumors. Loss of Kdm6a in murine RAS-transformed fibroblasts led to increased growth in vivo associated with decreased T cell infiltration.

KDM6A Regulates Immune Response Genes in Multiple Myeloma.

The histone H3K27 demethylase KDM6A is a tumor suppressor in multiple cancers, including multiple myeloma (MM). We created isogenic MM cells disrupted for KDM6A and tagged the endogenous protein to facilitate genome wide studies. KDM6A binds genes associated with immune recognition and cytokine signaling. Most importantly, KDM6A binds and activates NLRC5 and CIITA encoding regulators of Major Histocompatibility Complex (MHC) genes. Patient data indicate that NLRC5 and CIITA, are downregulated in MM with low KDM6A expression. Chromatin analysis shows that KDM6A binds poised and active enhancers and KDM6A loss led to decreased H3K27ac at enhancers, increased H3K27me3 levels in body of genes bound by KDM6A and decreased gene expression. Reestablishing histone acetylation with an HDAC3 inhibitor leads to upregulation of MHC expression, offering a strategy to restore immunogenicity of KDM6A deficient tumors. Loss of Kdm6a in murine RAS-transformed fibroblasts led to increased growth in vivo associated with decreased T cell infiltration. Statement of significance: We show that KDM6A participates in immune recognition of myeloma tumor cells by directly regulating the expression of the master regulators of MHC-I and II, NLRC5 and CIITA. The expression of these regulators can by rescued by the HDAC3 inhibitors in KDM6A-null cell lines.

Hematopoietic-specific heterozygous loss of Dnmt3a exacerbates colitis-associated colon cancer.

Clonal hematopoiesis (CH) is defined as clonal expansion of mutant hematopoietic stem cells absent diagnosis of a hematologic malignancy. Presence of CH in solid tumor patients, including colon cancer, correlates with shorter survival. We hypothesized that bone marrow-derived cells with heterozygous loss-of-function mutations of DNMT3A, the most common genetic alteration in CH, contribute to the pathogenesis of colon cancer. In a mouse model that combines colitis-associated colon cancer (CAC) with experimental CH driven by Dnmt3a+/Δ, we found higher tumor penetrance and increased tumor burden compared with controls. Histopathological analysis revealed accentuated colonic epithelium injury, dysplasia, and adenocarcinoma formation. Transcriptome profiling of colon tumors identified enrichment of gene signatures associated with carcinogenesis, including angiogenesis. Treatment with the angiogenesis inhibitor axitinib eliminated the colon tumor-promoting effect of experimental CH driven by Dnmt3a haploinsufficiency and rebalanced hematopoiesis. This study provides conceptually novel insights into non-tumor-cell-autonomous effects of hematopoietic alterations on colon carcinogenesis and identifies potential therapeutic strategies.

PRC2 Inhibitors Overcome Glucocorticoid Resistance Driven by NSD2 Mutation in Pediatric Acute Lymphoblastic Leukemia.

Mutations in epigenetic regulators are common in relapsed pediatric acute lymphoblastic leukemia (ALL). Here, we uncovered the mechanism underlying the relapse of ALL driven by an activating mutation of the NSD2 histone methyltransferase (p.E1099K). Using high-throughput drug screening, we found that NSD2-mutant cells were specifically resistant to glucocorticoids. Correction of this mutation restored glucocorticoid sensitivity. The transcriptional response to glucocorticoids was blocked in NSD2-mutant cells due to depressed glucocorticoid receptor (GR) levels and the failure of glucocorticoids to autoactivate GR expression. Although H3K27me3 was globally decreased by NSD2 p.E1099K, H3K27me3 accumulated at the NR3C1 (GR) promoter. Pretreatment of NSD2 p.E1099K cell lines and patient-derived xenograft samples with PRC2 inhibitors reversed glucocorticoid resistance in vitro and in vivo. PRC2 inhibitors restored NR3C1 autoactivation by glucocorticoids, increasing GR levels and allowing GR binding and activation of proapoptotic genes. These findings suggest a new therapeutic approach to relapsed ALL associated with NSD2 mutation. SIGNIFICANCE: NSD2 histone methyltransferase mutations observed in relapsed pediatric ALL drove glucocorticoid resistance by repression of the GR and abrogation of GR gene autoactivation due to accumulation of K3K27me3 at its promoter. Pretreatment with PRC2 inhibitors reversed resistance, suggesting a new therapeutic approach to these patients with ALL.This article is highlighted in the In This Issue feature, p. 1.

DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks.

PURPOSE: In acute myeloid leukemia (AML), recurrent DNA methyltransferase 3A (DNMT3A) mutations are associated with chemoresistance and poor prognosis, especially in advanced-age patients. Gene-expression studies in DNMT3A-mutated cells identified signatures implicated in deregulated DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here, we tested whether pharmacologically induced replication fork stalling, such as with cytarabine, creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. EXPERIMENTAL DESIGN: Leukemia cell lines, genetic mouse models, and isogenic cells with and without DNMT3A(mut) were used to evaluate sensitivity to nucleoside analogues such as cytarabine in vitro and in vivo, followed by analysis of DNA damage and signaling, replication restart, and cell-cycle progression on treatment and after drug removal. Transcriptome profiling identified pathways deregulated by DNMT3A(mut) expression. RESULTS: We found increased sensitivity to pharmacologically induced replication stress in cells expressing DNMT3A(R882)-mutant, with persistent intra-S-phase checkpoint activation, impaired PARP1 recruitment, and elevated DNA damage, which was incompletely resolved after drug removal and carried through mitosis. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine washout demonstrated a higher rate of fork collapse in DNMT3A(mut)-expressing cells. RNA-seq studies supported deregulated cell-cycle progression and p53 activation, along with splicing, ribosome biogenesis, and metabolism. CONCLUSIONS: Together, our studies show that DNMT3A mutations underlie a defect in recovery from replication fork arrest with subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during replication stress. See related commentary by Viny, p. 573.

Healthcare Utilization Patterns Among Children With a History of Child Protective Services Investigations.

PURPOSE: Examine whether children with a prior child protective services (CPS) investigation had different healthcare utilization compared to children without a history of CPS investigations. METHODS: The Children's Health Assessment and Planning Survey assessed 6,492 primary caregivers of children ages 0-17 years residing in North Texas in 2015. Caregivers reported prior CPS investigations and child healthcare utilization (emergency department [ED] use, unmet medication needs, and unmet medical care needs). PRINCIPLE FINDINGS: A total of 408 (5%) caregivers reported their child had a CPS investigation. Children with CPS investigations had greater odds of visiting the ED (OR = 1.9; 95% CI: 1.4, 2.5) and not receiving necessary medical care (OR = 1.9; 95% CI: 1.4, 2.8) compared to children without a CPS investigation. CONCLUSIONS: Prior CPS investigation was associated with disparities in receipt of necessary medical care and ED utilization for children.

Urinary Imaging Findings in Young Infants With Bacteremic Urinary Tract Infection.

OBJECTIVES: To describe renal ultrasound (RUS) and voiding cystourethrogram (VCUG) findings and determine predictors of abnormal imaging in young infants with bacteremic urinary tract infection (UTI). METHODS: We used retrospective data from a multicenter sample of infants younger than 3 months with bacteremic UTI, defined as the same pathogenic organism in blood and urine. Infants were excluded if they had any major comorbidities, known urologic abnormalities at time of presentation, required intensive unit care, or had no imaging performed. Imaging results as stated in the radiology reports were categorized by a pediatric urologist. RESULTS: Of the 276 infants, 19 were excluded. Of the remaining 257 infants, 254 underwent a RUS and 224 underwent a VCUG. Fifty-five percent had ≥1 RUS abnormalities. Thirty-four percent had ≥1 VCUG abnormalities, including vesicoureteral reflux (VUR, 27%), duplication (1.3%), and infravesicular abnormality (0.9%). Age <1 month, male sex, and non-Escherichia coli organism predicted an abnormal RUS, but only non-E coli organism predicted an abnormal VCUG. Seventeen of 96 infants (17.7%) with a normal RUS had an abnormal VCUG: 15 with VUR (Grade I-III = 13, Grade IV = 2), 2 with elevated postvoid residual, and 1 with infravesical abnormality. CONCLUSIONS: Although RUS and VCUG abnormalities were common in this cohort, the frequency and severity were similar to previous studies of infants with UTIs in general. Our findings do not support special consideration of bacteremia in imaging decisions for otherwise well-appearing young infants with UTI.

Bacteraemic urinary tract infection: management and outcomes in young infants.

OBJECTIVES: To determine predictors of parenteral antibiotic duration and the association between parenteral treatment duration and relapses in infants <3 months with bacteraemic urinary tract infection (UTI). DESIGN: Multicentre retrospective cohort study. SETTING: Eleven healthcare institutions across the USA. PATIENTS: Infants <3 months of age with bacteraemic UTI, defined as the same pathogenic organism isolated from blood and urine. MAIN OUTCOME MEASURES: Duration of parenteral antibiotic therapy, relapsed UTI within 30 days. RESULTS: The mean (±SD) duration of parenteral antibiotics for the 251 included infants was 7.8 days (±4 days), with considerable variability between institutions (mean range 5.5-12 days). Independent predictors of the duration of parenteral antibiotic therapy included (coefficient, 95% CI): age (-0.2 days, -0.3 days to -0.08 days, for each week older), year treated (-0.2 days, -0.4 to -0.03 days for each subsequent calendar year), male gender (0.9 days, 0.01 to 1.8 days), a positive repeat blood culture during acute treatment (3.5 days, 1.2-5.9 days) and a non-Escherichia coli organism (2.2 days, 0.8-3.6 days). No infants had a relapsed bacteraemic UTI. Six infants (2.4%) had a relapsed UTI (without bacteraemia). The duration of parenteral antibiotics did not differ between infants with and without a relapse (8.2 vs 7.8 days, p=0.81). CONCLUSIONS: Parenteral antibiotic treatment duration in young infants with bacteraemic UTI was variable and only minimally explained by measurable patient factors. Relapses were rare and were not associated with treatment duration. Shorter parenteral courses may be appropriate in some infants.

Diagnosis and management of bacteremic urinary tract infection in infants.

OBJECTIVES: To report the prevalence of bacteremia by age in a sample of infants<1 year of age with urinary tract infections (UTIs), to compare characteristics of infants with UTIs with and without bacteremia, and to describe treatment courses and 30-day outcomes in infants with UTIs with and without bacteremia. METHODS: We used a retrospective cross-sectional design to determine the prevalence of bacteremia in infants with UTIs at our institution. A double cohort design matching for age and gender was used to compare clinical characteristics and outcomes between infants with bacteremic versus nonbacteremic UTIs. RESULTS: We identified 1379 UTIs, with blood cultures obtained in 52% of cases. The prevalence of bacteremia was 4.1% (95% confidence interval 3.1%-5.3%) for all UTIs and 8% (95% confidence interval 6.1%-10.2%) for UTIs in which blood culture was obtained. Fifty-five infants with bacteremic UTIs were compared with 110 infants with nonbacteremic UTIs. Except for minor differences in the urinalysis and serum band count, there were no significant differences in clinical presentation between the 2 groups. Bacteremic infants received longer parenteral treatment courses than nonbacteremic infants (mean 6.7 vs 2.4 days, P<.001). Treatment courses in the bacteremic group were variable and predicted by age but not severity of illness. No bacteremic infant had recurrent UTI or bacteremia with the same organism within 30 days of discharge. CONCLUSIONS: Treatment was variable but outcomes were excellent in infants with bacteremic UTIs.

Legislative advocacy: evaluation of a grand rounds intervention for pediatricians.

OBJECTIVE: To evaluate the impact of a Grand Rounds Action Alert (GRAA) intervention on the behaviors, knowledge, and attitudes of pediatric grand rounds (GR) attendees; and to assess its acceptability. METHODS: A cross-sectional, quasi-experimental study was performed at a freestanding children's hospital. GRAA on child health legislative topics were presented in the first 2 minutes of the pediatric GR session as well as posted outside. Each session included an action item, such as writing/signing letters to elected officials or informational sheets with legislator contact information. Main outcome measures included self-reported behavior, advocacy knowledge, attitudes, and acceptability. RESULTS: One year after GRAA implementation, GR attendees with high exposure to the intervention were more likely to have written/signed a letter to a legislator compared to those with low/no exposure (60% vs 35%, P = .016). Those with high exposure were also more knowledgeable regarding financing of health care for low-income children (20% vs 5%, P = .027). Attitudes toward advocacy at baseline were positive: respondents agreed it is important to remain informed about (98%) and advocate for (94%) legislation favorable to children's health. Implementing this program was challenging, but the intervention was accepted favorably: 93% of respondents agreed that GRAA should continue. CONCLUSIONS: GRAA facilitated participation in legislative advocacy behaviors while improving self-perceived knowledge of legislative issues relating to children's health. They were well received in a large tertiary children's hospital.