Faculty Bibliography

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-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 shRNAs 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 to 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 p.E1099K mutation leads to drug resistance and a clonal advantage in childhood B-ALL.

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.

Favorable histology (FH) Wilms tumor (WT) is one of the most curable of all human cancers, yet a small minority of patients fail treatment. The underlying biological pathways that lead to therapy resistance are unknown. We report a case of initially unresectable, FH WT which revealed limited necrosis and persistent blastemal predominant histology following neoadjuvant chemotherapy. Despite intensification of therapy and whole abdominal radiation, the patient relapsed and succumbed to her disease. In an effort to discover candidate drivers of drug resistance, whole exome sequencing and copy number analysis were performed on samples from all 3 tumor specimens. Sequencing results revealed outgrowth of clones with a dramatically different genetic landscape including dominant mutations that could explain therapy evasion, some of which have not been previously reported in WT. Our results implicate PPM1D, previously shown to be associated with drug resistance in other tumors, as the major driver of treatment failure.

Background: Outcomes for children with acute lymphoblastic leukemia (ALL) have dramatically improved, but survival for patients who relapse remains poor. Mutations in genes encoding epigenetic modifiers are present in the majority of patients at relapse. In particular, activating mutations in NSD2 (MMSET, WHSC1), namely the glutamate to lysine substitution at amino acid 1099 (p.E1099K), are among the most common such mutations in epigenetic regulators. NSD2 converts histone 3 lysine 36 (H3K36) into its dimethylated form (H3K36me2) which in turn leads to stereotactic inhibition of EZH2 mediated H3K27me3. We and others have established that this leads to changes in chromatin state and gene expression. However, the pathways by which this leads to a clonal advantage remains elusive. Design/Method: We previously reported that overexpression of wild-type (WT) and p.E1099K mutant (EK) NSD2 in B-ALL cell lines led to unique cell context specific chromatin alterations and altered gene expression but did not lead to changes in proliferation or intrinsic drug resistance in vitro (Pierro et. al. Blood 2017 130:2474). We reasoned that these observations could be explained by the need for cooperating pathways that together with NSD2 EK lead to a clonal advantage. Thus we modulated expression of NSD2 using short hairpin RNAs (shRNAs) in the B-ALL cell line RS4;11 which harbors a heterozygous NSD2 EK mutation (NSD2 low). As a control, RS4;11 was also stably transduced with a non-targeting shRNA sequence (NSD2 high). Knockdown of NSD2 as well as decrease in H3K36me2 in NSD2 low lines was confirmed by Western Blot. Differences in gene regulation in NSD2 low cells were assessed by ChIPseq for CTCF, H3K9Ac, H3K27Ac, H3K36me2 and H3K27me3, and the results were correlated with RNAseq data. This data was then compared to RNAseq and ChIPseq data from REH and 697 NSD2 WT and EK overexpression cell lines in an effort to identify candidate genes or pathways preferentially regulated by the NSD2 EK mutation.
Result(s): NSD2 low cells displayed a distinct gene expression profile compared to NSD2 high with 301 upregulated and 573 downregulated genes (LFC 0.58, P = 0.05). When compared to gene expression data from our previously reported NSD2 overexpression cell lines, there was minimal overlap across cell lines with only 15 differentially expressed genes shared between RS4;11 NSD2 knockdown and REH EK overexpression cell lines and only 24 genes shared between RS4;11 NSD2 knockdown and 697 EK overexpression cell lines. Across all cell lines (RS4;11, REH and 697), only three genes (NSD2, SCN8 and PCNXL2) overlapped, all of which were upregulated in NSD2 high cell lines. Using less stringent criteria (LFC 0.26, P = 0.1), we observed greater overlap with 34 shared up and downregulated genes among lines. Of the shared genes, only ZNF521 which is overexpressed in NSD2 high cell lines, is known play a role in leukemogenesis. Moreover, RS4;11 pathway analysis revealed several biologically relevant pathways modulated by the NSD2 EK mutation such as Ras, integrin signaling, cholesterol/steroid biosynthesis, apoptosis and cell proliferation. Significant differences were also observed across epigenetic marks between RS 4;11 NSD2 high and low cells. In accordance with previously published data, we observed a global decrease in the H3K36me2 mark in RS4;11 NSD2 low lines. When aligned with changes in histone marks, among genes downregulated in NSD2 low cells there was a clear correlation with acquisition of the repressive H3K27me3 mark (and a decrease in the H3K9Ac mark). However among genes upregulated in NSD2 low cells we saw paradoxical increases in the H3K36me2 mark and decreases in the H3K27me3. Furthermore, gene expression was also influenced by marks not directly regulated by NSD2, namely H3K27ac and H3K9ac, indicating that local NSD2 mediated epigenetic changes are not the sole regulator of gene expression.
Conclusion(s): The activating p.E1099K substitution in NSD2 leads to a distinct gene expression profile in B-ALL cell lines that is cell context dependent. Moreover, while there is significant overlap in the transcriptional profile between WT and EK overexpression, there are distinct differences possibly indicating novel properties of the pE1099K substitution beyond enzyme hyperactivation. Our findings also imply that NSD2 EK collaborates with other leukemia associated alterations that result in clonal selection

OBJECTIVE:To determine reasons potentially amenable to interventions that mothers choose to supplement breastfeeding with formula in the immediate postpartum period. STUDY DESIGN:We distributed surveys to all mothers in the postpartum unit who delivered a live newborn on day of maternal discharge to assess feeding behaviors during their inpatient admission. We evaluated, when applicable, their reasons for supplementation and examined cultural and demographic information to uncover trends for formula use and potential areas for provider intervention. RESULTS:Seven hundred twelve of 1,400 mothers responded, of which 478 (65%) reported supplementing breastfeeding with formula (BF+F). The most common reasons for formula supplementation were perception of inadequate milk supply (36.4%), desire for sleep (35.4%), and a plan to breast and bottle feed (35.2%). Exclusive breastfeeding (EBF) was associated with primiparous status (OR 1.95; 95% CI 1.3-3.0), higher education level (OR 2.6; 95% CI 1.7-3.9), and having been breastfed as an infant (OR 1.54; 95% CI 1-2.37). Mothers who experienced skin-to-skin contact also had higher rates of EBF (29.5% versus 19.9%). Factors associated with exclusive formula feeding included single marital status, birth of mother in the United States, Catholic religion, multiparity, and cesarean delivery. Religious and cultural factors also played important roles in maternal feeding behaviors. CONCLUSION:Clinicians can anticipate risk factors for formula use in mothers who plan to breastfeed and tailor counseling appropriately to increase EBF rates.