CRISPR/Cas9 deletion of MIR155HG in human T cells reduces incidence and severity of acute GVHD in a xenogeneic model.

ABSTRACT: Acute graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic cell transplantation (allo-HCT). Using preclinical mouse models of disease, previous work in our laboratory has linked microRNA-155 (miR-155) to the development of acute GVHD. Transplantation of donor T cells from miR-155 host gene (MIR155HG) knockout mice prevented acute GVHD in multiple murine models of disease while maintaining critical graft-versus-leukemia (GVL) response, necessary for relapse prevention. In this study, we used clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 genome editing to delete miR-155 in primary T cells (MIR155HGΔexon3) from human donors, resulting in stable and sustained reduction in expression of miR-155. Using the xenogeneic model of acute GVHD, we show that NOD/SCID/IL2rγnull (NSG) mice receiving MIR155HGΔexon3 human T cells provide protection from lethal acute GVHD compared with mice that received human T cells with intact miR-155. MIR155HGΔexon3 human T cells persist in the recipients displaying decreased proliferation potential, reduced pathogenic T helper-1 cell population, and infiltration into GVHD target organs, such as the liver and skin. Importantly, MIR155HGΔexon3 human T cells retain GVL response significantly improving survival in an in vivo model of xeno-GVL. Altogether, we show that CRISPR/Cas9-mediated deletion of MIR155HG in primary human donor T cells is an innovative approach to generate allogeneic donor T cells that provide protection from lethal GVHD while maintaining robust antileukemic response.

Phase 1 study of selinexor in combination with salvage chemotherapy in Adults with relapsed or refractory Acute myeloid leukemia.

Selinexor, an oral inhibitor of the nuclear transport protein Exportin-1, shows promising single-agent activity in clinical trials of relapsed/refractory (R/R) acute myeloid leukemia (AML) and preclinical synergy with topoisomerase (topo) IIα inhibitors. We conducted a phase 1, dose-escalation study of selinexor with mitoxantrone, etoposide, and cytarabine (MEC) in 23 patients aged < 60 years with R/R AML. Due to dose-limiting hyponatremia in 2 patients on dose level 2 (selinexor 40 mg/m2), the maximum tolerated dose was 30 mg/m2. The most common grade ≥ 3 treatment-related non-hematologic toxicities were febrile neutropenia, catheter-related infections, diarrhea, hyponatremia, and sepsis. The overall response rate was 43% with 6 patients (26%) achieving complete remission (CR), 2 (9%) with CR with incomplete count recovery, and 2 (9%) with a morphologic leukemia-free state. Seven of 10 responders proceeded to allogeneic stem cell transplantation. The combination of selinexor with MEC is a feasibile treatment option for patients with R/R AML.

Epidermal growth factor-like 7 is a novel therapeutic target in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive, noncurative, mature B-cell lymphoma, with a median overall survival of 6-7 years. This underlines a need for effective therapeutic strategies to treat MCL better. Epidermal growth factor-like 7 (EGFL7) is a protein secreted by endothelial cells shown to play a critical role in angiogenesis. Our laboratory has previously demonstrated that EGFL7 supports the growth of leukemic blasts in patients with acute myeloid leukemia (AML); however, its role in MCL has not been investigated yet. In this study, we report that EGFL7 messenger RNA (mRNA) is increased in the cells of patients with MCL compared with cells from healthy controls, and patients with high EGFL7 are associated with lower overall survival rates. Furthermore, EGFL7 is increased in the plasma of patients with MCL compared with the plasma from healthy controls. We further show that EGFL7 binds to epidermal growth factor receptor (EGFR) and activates AKT signaling pathway in MCL cells and that blocking EGFL7 in MCL in patient and cell lines decreases cell growth and increases apoptosis in vitro. Finally, anti-EGFL7 treatment inhibits tumor size and prolongs survival in a mouse model of MCL. In conclusion, our study reveals a role for EGFL7 in MCL cell proliferation and highlights EGFL7 inhibition as a promising new treatment for patients with MCL.

Circular RNAs Could Encode Unique Proteins and Affect Cancer Pathways.

circRNAs constitute a novel class of RNA, generally considered as non-coding RNAs; nonetheless, their coding potential has been under scrutiny. In this work, we systematically explored the predicted proteins of more than 160,000 circRNAs detected by exome capture RNA-sequencing and collected in the MiOncoCirc pan-cancer compendium, including normal and cancer samples from different types of tissues. For the functional evaluation, we compared their primary structure and domain composition with those derived from the same linear mRNAs. Among the 4362 circRNAs potentially encoding proteins with a unique primary structure and 1179 encoding proteins with a novel domain composition, 183 were differentially expressed in cancer. In particular, eight were associated with prognosis in acute myeloid leukemia. The functional classification of the dysregulated circRNA-encoded polypeptides showed an enrichment in the heme and cancer signaling, DNA-binding, and phosphorylation processes, and disclosed the roles of some circRNA-based effectors in cancer.

Non-Coding RNAs Are Implicit in Chronic Myeloid Leukemia Therapy Resistance.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm initiated by the presence of the fusion gene BCR::ABL1. The development of tyrosine kinase inhibitors (TKIs) highly specific to p210BCR-ABL1, the constitutively active tyrosine kinase encoded by BCR::ABL1, has greatly improved the prognosis for CML patients. Now, the survival rate of CML nearly parallels that of age matched controls. However, therapy resistance remains a persistent problem in the pursuit of a cure. TKI resistance can be attributed to both BCR::ABL1 dependent and independent mechanisms. Recently, the role of non-coding RNAs (ncRNAs) has been increasingly explored due to their frequent dysregulation in a variety of malignancies. Specifically, microRNAs (miRNAs), circular RNAs (circRNAs), and long non-coding RNAs (lncRNAs) have been shown to contribute to the development and progression of therapy resistance in CML. Since each ncRNA exhibits multiple functions and is capable of controlling gene expression, they exert their effect on CML resistance through a diverse set of mechanisms and pathways. In most cases ncRNAs with tumor suppressing functions are silenced in CML, while those with oncogenic properties are overexpressed. Here, we discuss the relevance of many aberrantly expressed ncRNAs and their effect on therapy resistance in CML.

Modulating endothelial cells with EGFL7 to diminish aGVHD after allogeneic bone marrow transplantation in mice.

Acute graft-versus-host disease (aGVHD) is the second most common cause of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT), underscoring the need for novel therapies. Based on previous work that endothelial cell dysfunction is present in aGVHD and that epidermal growth factor-like domain 7 (EGFL7) plays a significant role in decreasing inflammation by repressing endothelial cell activation and T-cell migration, we hypothesized that increasing EGFL7 levels after allo-HSCT will diminish the severity of aGVHD. Here, we show that treatment with recombinant EGFL7 (rEGFL7) in 2 different murine models of aGVHD decreases aGVHD severity and improves survival in recipient mice after allogeneic transplantation with respect to controls without affecting graft-versus-leukemia effect. Furthermore, we showed that rEGFL7 treatment results in higher thymocytes, T, B, and dendritic cell counts in recipient mice after allo-HSCT. This study constitutes a proof of concept of the ability of rEGFL7 therapy to reduce GHVD severity and mortality after allo-HSCT.

Clinical and molecular relevance of genetic variants in the non-coding transcriptome of patients with cytogenetically normal acute myeloid leukemia.

Expression levels of long non-coding RNA (lncRNA) have been shown to associate with clinical outcome of patients with cytogenetically normal acute myeloid leukemia (CN-AML). However, the frequency and clinical significance of genetic variants in the nucleotide sequences of lncRNA in AML patients is unknown. Herein, we analyzed total RNA sequencing data of 377 younger adults (aged <60 years) with CN-AML, who were comprehensively characterized with regard to clinical outcome. We used available genomic databases and stringent filters to annotate genetic variants unequivocally located in the non-coding transcriptome of AML patients. We detected 981 variants, which are recurrently present in lncRNA that are expressed in leukemic blasts. Among these variants, we identified a cytosine-to-thymidine variant in the lncRNA RP5-1074L1.4 and a cytosine-to-thymidine variant in the lncRNA SNHG15, which independently associated with longer survival of CN-AML patients. The presence of the SNHG15 cytosine-to-thymidine variant was also found to associate with better outcome in an independent dataset of CN-AML patients, despite differences in treatment protocols and RNA sequencing techniques. In order to gain biological insights, we cloned and overexpressed both wild-type and variant versions of the SNHG15 lncRNA. In keeping with its negative prognostic impact, overexpression of the wild-type SNHG15 associated with higher proliferation rate of leukemic blasts when compared with the cytosine-to-thymidine variant. We conclude that recurrent genetic variants of lncRNA that are expressed in the leukemic blasts of CN-AML patients have prognostic and potential biological significance.

Molecular, clinical, and prognostic implications of PTPN11 mutations in acute myeloid leukemia.

Prognostic factors associated with chemotherapy outcomes in patients with acute myeloid leukemia (AML) are extensively reported, and one gene whose mutation is recognized as conferring resistance to several newer targeted therapies is protein tyrosine phosphatase non-receptor type 11 (PTPN11). The broader clinical implications of PTPN11 mutations in AML are still not well understood. The objective of this study was to determine which cytogenetic abnormalities and gene mutations co-occur with PTPN11 mutations and how PTPN11 mutations affect outcomes of patients treated with intensive chemotherapy. We studied 1725 patients newly diagnosed with AML (excluding acute promyelocytic leukemia) enrolled onto the Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology trials. In 140 PTPN11-mutated patient samples, PTPN11 most commonly co-occurred with mutations in NPM1, DNMT3A, and TET2. PTPN11 mutations were relatively common in patients with an inv(3)(q21q26)/t(3;3)(q21;q26) and a normal karyotype but were very rare in patients with typical complex karyotype and core-binding factor AML. Mutations in the N-terminal SH2 domain of PTPN11 were associated with a higher early death rate than those in the phosphatase domain. PTPN11 mutations did not affect outcomes of NPM1-mutated patients, but these patients were less likely to have co-occurring kinase mutations (ie, FLT3-ITD), suggesting activation of overlapping signaling pathways. However, in AML patients with wild-type NPM1, PTPN11 mutations were associated with adverse patient outcomes, providing a rationale to study the biology and treatment approaches in this molecular group. This trial was registered at www.clinicaltrials.gov as #NCT00048958 (CALGB 8461), #NCT00899223 (CALGB 9665), and #NCT00900224 (CALGB 20202).

Clinical Applications of MicroRNAs in Acute Myeloid Leukemia: A Mini-Review.

MicroRNAs (miRs) are short non-coding RNAs, typically 18-25 nucleotides in length, that are critically important, through their direct effects on target mRNAs, in a variety of cellular processes including cell differentiation, proliferation and survival. Dysregulated miR expression has been identified in numerous cancer types including acute myeloid leukemia (AML). From a clinical standpoint, several miRs have been shown to associate with prognosis in AML patients. Furthermore, they also carry the potential to be used as biomarkers and to inform medical decision making. In addition, several preclinical studies have provided strong rationale to develop novel therapeutic strategies to target miRs in AML. This review will focus on potential clinical applications of miRs in adult AML and will discuss unique miR signatures in specific AML subtypes, their role in prognostication and response to therapy, as well as miRs that are promising therapeutic targets and ongoing clinical trials directed towards targeting clinically relevant miRs in AML that could allow for improvements in current treatment strategies.

Precision oncology in AML: validation of the prognostic value of the knowledge bank approach and suggestions for improvement.

Recently, a novel knowledge bank (KB) approach to predict outcomes of individual patients with acute myeloid leukemia (AML) was developed using unbiased machine learning. To validate its prognostic value, we analyzed 1612 adults with de novo AML treated on Cancer and Leukemia Group B front-line trials who had pretreatment clinical, cytogenetics, and mutation data on 81 leukemia/cancer-associated genes available. We used receiver operating characteristic (ROC) curves and the area under the curve (AUC) to evaluate the predictive values of the KB algorithm and other risk classifications. The KB algorithm predicted 3-year overall survival (OS) probability in the entire patient cohort (AUCKB = 0.799), and both younger (< 60 years) (AUCKB = 0.747) and older patients (AUCKB = 0.770). The KB algorithm predicted non-remission death (AUCKB = 0.860) well but was less accurate in predicting relapse death (AUCKB = 0.695) and death in first complete remission (AUCKB = 0.603). The KB algorithm's 3-year OS predictive value was higher than that of the 2017 European LeukemiaNet (ELN) classification (AUC2017ELN = 0.707, p < 0.001) and 2010 ELN classification (AUC2010ELN = 0.721, p < 0.001) but did not differ significantly from that of the 17-gene stemness score (AUC17-gene = 0.732, p = 0.10). Analysis of additional cytogenetic and molecular markers not included in the KB algorithm revealed that taking into account atypical complex karyotype, infrequent recurrent balanced chromosome rearrangements and mutational status of the SAMHD1, AXL and NOTCH1 genes may improve the KB algorithm. We conclude that the KB algorithm has a high predictive value that is higher than those of the 2017 and 2010 ELN classifications. Inclusion of additional genetic features might refine the KB algorithm.

Methods Used to Make Lipid Nanoparticles to Deliver LNA Gapmers Against lncRNAs into Acute Myeloid Leukemia (AML) Blasts.

Developing strategies to target lncRNAs are needed. In this chapter, we describe in detail a method to deliver antisense oligonucleotides into acute myeloid leukemia cells using lipid nanoparticles tagged with the transferrin receptor. While this chapter is focused on the delivery method, we also discuss important considerations about the design of antisense oligonucleotides (ASOs). The strategy described here has been used successfully to deliver ASOs into leukemic blasts and stem cells.

ATF3 coordinates serine and nucleotide metabolism to drive cell cycle progression in acute myeloid leukemia.

Metabolic reprogramming is a common feature of many human cancers, including acute myeloid leukemia (AML). However, the upstream regulators that promote AML metabolic reprogramming and the benefits conferred to leukemia cells by these metabolic changes remain largely unknown. We report that the transcription factor ATF3 coordinates serine and nucleotide metabolism to maintain cell cycling, survival, and the differentiation blockade in AML. Analysis of mouse and human AML models demonstrate that ATF3 directly activates the transcription of genes encoding key enzymatic regulators of serine synthesis, one-carbon metabolism, and de novo purine and pyrimidine synthesis. Total steady-state polar metabolite and heavy isotope tracing analyses show that ATF3 inhibition reduces de novo serine synthesis, impedes the incorporation of serine-derived carbons into newly synthesized purines, and disrupts pyrimidine metabolism. Importantly, exogenous nucleotide supplementation mitigates the anti-leukemia effects of ATF3 inhibition. Together, these findings reveal the dependence of AML on ATF3-regulated serine and nucleotide metabolism.

DNA methylation epitypes highlight underlying developmental and disease pathways in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a molecularly complex disease characterized by heterogeneous tumor genetic profiles and involving numerous pathogenic mechanisms and pathways. Integration of molecular data types across multiple patient cohorts may advance current genetic approaches for improved subclassification and understanding of the biology of the disease. Here, we analyzed genome-wide DNA methylation in 649 AML patients using Illumina arrays and identified a configuration of 13 subtypes (termed "epitypes") using unbiased clustering. Integration of genetic data revealed that most epitypes were associated with a certain recurrent mutation (or combination) in a majority of patients, yet other epitypes were largely independent. Epitypes showed developmental blockage at discrete stages of myeloid differentiation, revealing epitypes that retain arrested hematopoietic stem-cell-like phenotypes. Detailed analyses of DNA methylation patterns identified unique patterns of aberrant hyper- and hypomethylation among epitypes, with variable involvement of transcription factors influencing promoter, enhancer, and repressed regions. Patients in epitypes with stem-cell-like methylation features showed inferior overall survival along with up-regulated stem cell gene expression signatures. We further identified a DNA methylation signature involving STAT motifs associated with FLT3-ITD mutations. Finally, DNA methylation signatures were stable at relapse for the large majority of patients, and rare epitype switching accompanied loss of the dominant epitype mutations and reversion to stem-cell-like methylation patterns. These results show that DNA methylation-based classification integrates important molecular features of AML to reveal the diverse pathogenic and biological aspects of the disease.

Gene expression signature predicts relapse in adult patients with cytogenetically normal acute myeloid leukemia.

Although ∼80% of adult patients with cytogenetically normal acute myeloid leukemia (CN-AML) achieve a complete remission (CR), more than half of them relapse. Better identification of patients who are likely to relapse can help to inform clinical decisions. We performed RNA sequencing on pretreatment samples from 268 adults with de novo CN-AML who were younger than 60 years of age and achieved a CR after induction treatment with standard "7+3" chemotherapy. After filtering for genes whose expressions were associated with gene mutations known to impact outcome (ie, CEBPA, NPM1, and FLT3-internal tandem duplication [FLT3-ITD]), we identified a 10-gene signature that was strongly predictive of patient relapse (area under the receiver operating characteristics curve [AUC], 0.81). The signature consisted of 7 coding genes (GAS6, PSD3, PLCB4, DEXI, JMY, NRP1, C10orf55) and 3 long noncoding RNAs. In multivariable analysis, the 10-gene signature was strongly associated with relapse (P < .001), after adjustment for the FLT3-ITD, CEBPA, and NPM1 mutational status. Validation of the expression signature in an independent patient set from The Cancer Genome Atlas showed the signature's strong predictive value, with AUC = 0.78. Implementation of the 10-gene signature into clinical prognostic stratification could be useful for identifying patients who are likely to relapse.

Poor Survival and Differential Impact of Genetic Features of Black Patients with Acute Myeloid Leukemia.

Clinical outcome of patients with acute myeloid leukemia (AML) is associated with cytogenetic and molecular factors and patient demographics (e.g., age and race). We compared survival of 25,523 non-Hispanic Black and White adults with AML using Surveillance Epidemiology and End Results (SEER) Program data and performed mutational profiling of 1,339 patients with AML treated on frontline Alliance for Clinical Trials in Oncology (Alliance) protocols. Black patients had shorter survival than White patients, both in SEER and in the setting of Alliance clinical trials. The disparity was especially pronounced in Black patients <60 years, after adjustment for socioeconomic (SEER) and molecular (Alliance) factors. Black race was an independent prognosticator of poor survival. Gene mutation profiles showed fewer NPM1 and more IDH2 mutations in younger Black patients. Overall survival of younger Black patients was adversely affected by IDH2 mutations and FLT3-ITD, but, in contrast to White patients, was not improved by NPM1 mutations. SIGNIFICANCE: We show that young Black patients have not benefited as much as White patients from recent progress in AML treatment in the United States. Our data suggest that both socioeconomic factors and differences in disease biology contribute to the survival disparity and need to be urgently addressed.See related commentary by Vyas, p. 540.This article is highlighted in the In This Issue feature, p. 521.

Incidence of venous thrombosis after peg-asparaginase in adolescent and young adults with acute lymphoblastic leukemia.

AIM: There are limited data describing incidence of symptomatic venous thromboembolism (VTE) in adolescent and young adult (AYA) acute lymphoblastic leukemia (ALL) patients receiving peg-asparaginase. MATERIALS & METHODS: Single-institution retrospective analysis of 44 AYA ALL patients treated with peg-asparaginase. Rates of VTE and proposed risk factors were assessed. RESULTS: 18 patients (41%) had a symptomatic VTE following peg-asparaginase. The cumulative incidence rate was 25% (95% CI: 13-38%) within 30 days of the initial dose. Personal history of thrombosis was statistically significantly associated with an increased risk of VTE with HR of 2.73 (95% CI: 1.40-5.33, p = 0.003) after adjusting for gender. CONCLUSION: These data indicate a high rate of VTE in the AYA ALL population following treatment with peg-asparaginase.

Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions.

Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that MIR300 has antiproliferative and PP2A-activating functions that are dose dependently differentially induced by CCND2/CDK6 and SET inhibition, respectively. MIR300 is upregulated in CML LSCs and NK cells by bone marrow microenvironment (BMM) signals to induce quiescence and impair immune response, respectively. Conversely, BCR-ABL1 downregulates MIR300 in CML progenitors to prevent growth arrest and PP2A-mediated apoptosis. Quiescent LSCs escape apoptosis by upregulating TUG1 long noncoding RNA that uncouples and limits MIR300 function to cytostasis. Genetic and pharmacologic MIR300 modulation and/or PP2A-activating drug treatment restore NK cell activity, inhibit BMM-induced growth arrest, and selectively trigger LSC apoptosis in vitro and in patient-derived xenografts; hence, the importance of MIR300 and PP2A activity for CML development and therapy.