Purpose: Pediatric oncology patients undergoing active chemotherapy are suspected to be at a high risk for severe disease secondary to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection; however, data to support this are lacking. We aim to describe the characteristics of coronavirus disease 2019 (COVID-19) in this population and also its impact on pediatric cancer care in the New York region during the peak of the pandemic.

Whereas the improvement in outcome for children with acute lymphoblastic leukemia has been gratifying, the poor outcome of patients who relapse warrants novel treatment approaches. Previously, we identified a characteristic relapse-specific gene expression and methylation signature associated with chemoresistance using a large cohort of matched-diagnosis relapse samples. We hypothesized that "reversing" such a signature might restore chemosensitivity. In the present study, we demonstrate that the histone deacetylase inhibitor vorinostat not only reprograms the aberrant gene expression profile of relapsed blasts by epigenetic mechanisms, but is also synergistic when applied before chemotherapy in primary patient samples and leukemia cell lines. Furthermore, incorporation of the DNA methyltransferase inhibitor decitabine led to reexpression of genes shown to be preferentially methylated and silenced at relapse. Combination pretreatment with vorinostat and decitabine resulted in even greater cytotoxicity compared with each agent individually with chemotherapy. Our results indicate that acquisition of chemoresistance at relapse may be driven in part by epigenetic mechanisms. Incorporation of these targeted epigenetic agents to the standard chemotherapy backbone is a promising approach to the treatment of relapsed pediatric acute lymphoblastic leukemia. IntroductionRelapsed acute lymphoblastic leukemia (ALL) is one of the leading causes of death among children with cancer. A hallmark of relapsed blasts is their intrinsic chemoresistance compared with what is observed at initial diagnosis. 1,2 Given the frequent failure of conventional salvage chemotherapy, including intensified drug schedules and stem cell transplantation, in the treatment of relapsed ALL, 3,4 innovative strategies are urgently needed.In recent years, it has become clear that cancer can be driven by patterns of altered gene expression mediated not by mechanisms that affect the primary DNA sequence, but through the "epigenetic" processes of DNA-promoter methylation and histone modification. 5,6 DNA methylation is catalyzed by DNA methyltransferases (DNMTs) and has been shown to be an important contributor to carcinogenesis, acting by silencing tumor suppressor genes in many tumor types, including hematologic malignancies. 6,7 Chromatin structure is also regulated by the "histone code," which refers to posttranslational modifications (ie, methylation, acetylation, phosphorylation, and ubiquitination) of key lysine residues on core histone proteins. 8 These 2 epigenetic processes of DNA-promoter methylation and histone modifications are clearly interdependent and coordinated. [9][10][11][12] DNMT inhibitors such as 5-azacitidine and decitabine have the potential to reverse promoter hypermethylation in tumor cells, leading to reexpression of aberrantly silenced genes and inducing tumor cell death. 13 DNMT inhibitors have been demonstrated to be effective therapy for myelodysplastic syndrome, which is characterized by global promoter hypermethylation. 14...

• Genetic or pharmacologic inhibition of MEK4 and MEK2 enhances prednisoloneinduced cell death in ALL models.• MAPK signaling cascades are activated at relapse compared to diagnosis in ALL samples and have enhanced response to MEK inhibition.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. (Blood. 2015;126(19):2202-2212

Summary While childhood acute lymphoblastic leukemia (ALL) is now highly curable, the dismal prognosis for children who relapse warrants novel therapeutic approaches. Previously, using an integrated genomic analysis of matched diagnosis - relapse paired samples, we identified overactivation of the Wnt pathway as a possible mechanism of recurrence. To validate these findings and document whether Wnt inhibition may sensitize cells to chemotherapy, we analyzed the expression of Activated β-catenin (and its downstream target BIRC5) using multiparameter phosphoflow cytometry and tested the efficacy of a recently developed Wnt inhibitor, iCRT14, in ALL cell lines and patient samples. We observed increased activation of β-catenin at relapse in 6 /10 patients. Furthermore, treatment of leukemic cell lines with iCRT14 led to significant downregulation of Wnt target genes and combination with traditional chemotherapeutic drugs resulted in a synergistic decrease in viability as well as a significant increase in apoptotic cell death. Finally, pre-treatment of purified blasts from patients with relapsed leukemia with the Wnt inhibitor followed by exposure to prednisolone, restored chemosensitivity in these cells. Our results demonstrate that overactivation of the Wnt pathway may contribute to chemoresistance in relapsed childhood ALL and that Wnt-inhibition may be a promising therapeutic approach.

Although the cure rate of newly diagnosed acute lymphoblastic leukemia (ALL) has improved over the past four decades, the outcome for patients who relapse remains poor. New therapies are needed for these patients. Our previous global gene expression analysis in a series of paired diagnosis-relapse pediatric patient samples revealed that the antiapoptotic gene survivin was consistently upregulated upon disease relapse. In this study, we demonstrate a link between survivin expression and drug resistance and test the efficacy of a novel antisense agent in promoting apoptosis when combined with chemotherapy. Gene-silencing experiments targeting survivin mRNA using either short-hairpin RNA (shRNA) or a locked antisense oligonucleotide (LNA-ON) specifically reduced gene expression and induced apoptosis in leukemia cell lines. When used in combination with chemotherapy, the survivin shRNA and LNA-ON potentiated the chemotherapeutic antileukemia effect. Moreover, in a mouse primary xenograft model of relapse ALL, the survivin LNA-ON decreased survivin expression in a subset of animals, and produced a statistically significant decrease in tumor progression. Taken together, these findings suggest that targeting endogenous levels of survivin mRNA by LNA-ON methods may augment the response to standard chemotherapy by sensitizing otherwise resistant tumor cells to chemotherapy.