Heterogeneous nuclear ribonucleoprotein K is overexpressed in acute myeloid leukemia and causes myeloproliferation in mice via altered Runx1 splicing.

Acute myeloid leukemia (AML) is driven by numerous molecular events that contribute to disease progression. Herein, we identify hnRNP K overexpression as a recurrent abnormality in AML that negatively correlates with patient survival. Overexpression of hnRNP K in murine fetal liver cells results in altered self-renewal and differentiation potential. Further, murine transplantation models reveal that hnRNP K overexpression results in myeloproliferation in vivo. Mechanistic studies expose a direct functional relationship between hnRNP K and RUNX1-a master transcriptional regulator of hematopoiesis often dysregulated in leukemia. Molecular analyses show that overexpression of hnRNP K results in an enrichment of an alternatively spliced isoform of RUNX1 lacking exon 4. Our work establishes hnRNP K's oncogenic potential in influencing myelogenesis through its regulation of RUNX1 splicing and subsequent transcriptional activity.

Targeting the NRF2/HO-1 Antioxidant Pathway in FLT3-ITD-Positive AML Enhances Therapy Efficacy.

Acute myeloid leukemia (AML) is a molecularly heterogenous hematological malignancy, with one of the most common mutations being internal tandem duplication (ITD) of the juxtamembrane domain of the fms-like tyrosine kinase receptor-3 (FLT3). Despite the development of FLT3-directed tyrosine kinase inhibitors (TKI), relapse and resistance are problematic, requiring improved strategies. In both patient samples and cell lines, FLT3-ITD raises levels of reactive oxygen species (ROS) and elicits an antioxidant response which is linked to chemoresistance broadly in AML. NF-E2-related factor 2 (NRF2) is a transcription factor regulating the antioxidant response including heme oxygenase -1 (HO-1), a heat shock protein implicated in AML resistance. Here, we demonstrate that HO-1 is elevated in FLT3-ITD-bearing cells compared to FLT3-wild type (WT). Transient knockdown or inhibitor-based suppression of HO-1 enhances vulnerability to the TKI, quizartinib, in both TKI-resistant and sensitive primary AML and cell line models. NRF2 suppression (genetically or pharmacologically using brusatol) results in decreased HO-1, suggesting that TKI-resistance is dependent on an active NRF2-driven pathway. In AML-patient derived xenograft (PDX) models, brusatol, in combination with daunorubicin, reduces leukemia burden and prolongs survival. Cumulatively, these data encourage further development of brusatol and NRF2 inhibition as components of combination therapy for refractory AML.

AXL/MERTK inhibitor ONO-7475 potently synergizes with venetoclax and overcomes venetoclax resistance to kill FLT3-ITD acute myeloid leukemia.

FMS-like Tyrosine Kinase 3 (FLT3) mutation is associated with poor survival in acute myeloid leukemia (AML). The specific Anexelekto/MER Tyrosine Kinase (AXL) inhibitor, ONO-7475, kills FLT3-mutant AML cells with targets including Extracellular- signal Regulated Kinase (ERK) and Myeloid Cell Leukemia 1 (MCL1). ERK and MCL1 are known resistance factors for Venetoclax (ABT-199), a popular drug for AML therapy, prompting the investigation of the efficacy of ONO-7475 in combination with ABT-199 in vitro and in vivo. ONO-7475 synergizes with ABT-199 to potently kill FLT3-mutant acute myeloid leukemia cell lines and primary cells. ONO-7475 is effective against ABT-199-resistant cells including cells that overexpress MCL1. Proteomic analyses revealed that ABT-199-resistant cells expressed elevated levels of pro-growth and anti-apoptotic proteins compared to parental cells, and that ONO-7475 reduced the expression of these proteins in both the parental and ABT-199-resistant cells. ONO-7475 treatment significantly extended survival as a single in vivo agent using acute myeloid leukemia cell lines and PDX models. Compared to ONO-7474 monotherapy, the combination of ONO-7475/ABT-199 was even more potent in reducing leukemic burden and prolonging the survival of mice in both model systems. These results suggest that the ONO-7475/ABT-199 combination may be effective for AML therapy.

Prognostic and therapeutic implications of measurable residual disease in acute myeloid leukemia.

Quantification of measurable residual disease (MRD) provides critical prognostic information in acute myeloid leukemia (AML). A variety of platforms exist for MRD detection, varying in their sensitivity and applicability to individual patients. MRD detected by quantitative polymerase chain reaction, multiparameter flow cytometry, or next-generation sequencing has prognostic implications in various subsets of AML and at various times throughout treatment. While it is overwhelmingly evident that minute levels of remnant disease confer increased risk of relapse and shortened survival, the therapeutic implications of MRD remain less clear. The use of MRD as a guide to selecting the most optimal post-remission therapy, including hematopoietic stem cell transplant or maintenance therapy with hypomethylating agents, small molecule inhibitors, or immunotherapy is an area of active investigation. In addition, whether there are sufficient data to use MRD negativity as a surrogate endpoint in clinical trial development is controversial. In this review, we will critically examine the methods used to detect MRD, its role as a prognostic biomarker, MRD-directed therapeutics, and its potential role as a study endpoint.

Overexpression of CD200 is a Stem Cell-Specific Mechanism of Immune Evasion in AML.

BACKGROUND: Acute myeloid leukemia (AML) stem cells (LSCs) are capable of surviving current standard chemotherapy and are the likely source of deadly, relapsed disease. While stem cell transplant serves as proof-of-principle that AML LSCs can be eliminated by the immune system, the translation of existing immunotherapies to AML has been met with limited success. Consequently, understanding and exploiting the unique immune-evasive mechanisms of AML LSCs is critical. METHODS: Analysis of stem cell datasets and primary patient samples revealed CD200 as a putative stem cell-specific immune checkpoint overexpressed in AML LSCs. Isogenic cell line models of CD200 expression were employed to characterize the interaction of CD200+ AML with various immune cell subsets both in vitro and in peripheral blood mononuclear cell (PBMC)-humanized mouse models. CyTOF and RNA-sequencing were performed on humanized mice to identify novel mechanisms of CD200-mediated immunosuppression. To clinically translate these findings, we developed a fully humanized CD200 antibody (IgG1) that removed the immunosuppressive signal by blocking interaction with the CD200 receptor while also inducing a potent Fc-mediated response. Therapeutic efficacy of the CD200 antibody was evaluated using both humanized mice and patient-derived xenograft models. RESULTS: Our results demonstrate that CD200 is selectively overexpressed in AML LSCs and is broadly immunosuppressive by impairing cytokine secretion in both innate and adaptive immune cell subsets. In a PBMC-humanized mouse model, CD200+ leukemia progressed rapidly, escaping elimination by T cells, compared with CD200- AML. T cells from mice with CD200+ AML were characterized by an abundance of metabolically quiescent CD8+ central and effector memory cells. Mechanistically, CD200 expression on AML cells significantly impaired OXPHOS metabolic activity in T cells from healthy donors. Importantly, CD200 antibody therapy could eliminate disease in the presence of graft-versus-leukemia in immune competent mice and could significantly improve the efficacy of low-intensity azacitidine/venetoclax chemotherapy in immunodeficient hosts. CONCLUSIONS: Overexpression of CD200 is a stem cell-specific marker that contributes to immunosuppression in AML by impairing effector cell metabolism and function. CD200 antibody therapy is capable of simultaneously reducing CD200-mediated suppression while also engaging macrophage activity. This study lays the groundwork for CD200-targeted therapeutic strategies to eliminate LSCs and prevent AML relapse.

The Eµ-hnRNP K Murine Model of Lymphoma: Novel Insights into the Role of hnRNP K in B-Cell Malignancies.

B-cell lymphomas are one of the most biologically and molecularly heterogeneous group of malignancies. The inherent complexity of this cancer subtype necessitates the development of appropriate animal model systems to characterize the disease with the ultimate objective of identifying effective therapies. In this article, we discuss a new driver of B-cell lymphomas - hnRNP K (heterogenous nuclear ribonucleoprotein K)-an RNA-binding protein. We introduce the Eµ-Hnrnpk mouse model, a murine model characterized by hnRNP K overexpression in B cells, which develops B-cell lymphomas with high penetrance. Molecular analysis of the disease developed in this model reveals an upregulation of the c-Myc oncogene via post-transcriptional and translational mechanisms underscoring the impact of non-genomic MYC activation in B-cell lymphomas. Finally, the transplantability of the disease developed in Eµ-Hnrnpk mice makes it a valuable pre-clinical platform for the assessment of novel therapeutics.

Two Cases of Possible Familial Chronic Myeloid Leukemia in a Family with Extensive History of Cancer.

CML is defined by the presence of an oncogenic fusion protein caused by a reciprocal translocation between chromosomes 9q and 22q. While our molecular understanding of CML pathogenesis has revolutionized drug development for this disease, we have yet to identify many predisposing factors for CML. Familial occurrence of CML has been rarely reported. Here, we describe 2 cases of CML in a 24-year-old woman and in her 73-year-old maternal great aunt. We describe genetic variants in these patients and report on their environmental exposures that may have contributed to CML pathogenesis. The possible familial association of these 2 cases of CML warrants further investigation into more definitive etiologies of this disease.

Uncovering the Role of RNA-Binding Protein hnRNP K in B-Cell Lymphomas.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA-binding protein that is aberrantly expressed in cancers. We and others have previously shown that reduced hnRNP K expression downmodulates tumor-suppressive programs. However, overexpression of hnRNP K is the more commonly observed clinical phenomenon, yet its functional consequences and clinical significance remain unknown. METHODS: Clinical implications of hnRNP K overexpression were examined through immunohistochemistry on samples from patients with diffuse large B-cell lymphoma who did not harbor MYC alterations (n = 75). A novel transgenic mouse model that overexpresses hnRNP K specifically in B cells was generated to directly examine the role of hnRNP K overexpression in mice (three transgenic lines). Molecular consequences of hnRNP K overexpression were determined through proteomics, formaldehyde-RNA-immunoprecipitation sequencing, and biochemical assays. Therapeutic response to BET-bromodomain inhibition in the context of hnRNP K overexpression was evaluated in vitro and in vivo (n = 3 per group). All statistical tests were two-sided. RESULTS: hnRNP K is overexpressed in diffuse large B-cell lymphoma patients without MYC genomic alterations. This overexpression is associated with dismal overall survival and progression-free survival (P < .001). Overexpression of hnRNP K in transgenic mice resulted in the development of lymphomas and reduced survival (P < .001 for all transgenic lines; Line 171[n = 30]: hazard ratio [HR] = 64.23, 95% confidence interval [CI] = 26.1 to 158.0; Line 173 [n = 31]: HR = 25.27, 95% CI = 10.3 to 62.1; Line 177 [n = 25]: HR = 119.5, 95% CI = 42.7 to 334.2, compared with wild-type mice). Clinical samples, mouse models, global screening assays, and biochemical studies revealed that hnRNP K's oncogenic potential stems from its ability to posttranscriptionally and translationally regulate MYC. Consequently, Hnrnpk overexpression renders cells sensitive to BET-bromodomain-inhibition in both in vitro and transplantation models, which represents a strategy for mitigating hnRNP K-mediated c-Myc activation in patients. CONCLUSION: Our findings indicate that hnRNP K is a bona fide oncogene when overexpressed and represents a novel mechanism for c-Myc activation in the absence of MYC lesions.

Emerging treatment options for patients with p53-pathway-deficient CLL.

Over the past 40 years, p53 has been the most widely studied protein in cancer biology. Originally thought to be an oncogene due to its stabilization in many cancers, it is now considered to be one of the most critical tumor suppressors in a cell's ability to combat neoplastic transformation. Due to its critical roles in apoptosis, cell-cycle arrest, and senescence, TP53 deletions and mutations are commonly observed and are often a portent of treatment failures and poor clinical outcomes. This is particularly true in chronic lymphocytic leukemia (CLL), as patients with p53 alterations have historically had dismal outcomes. As such, the tremendous efforts made to better understand the functions of p53 in CLL have contributed substantially to recent advances in treating patients with p53-pathway-deficient CLL.

Antileukemia Effects of Notch-Mediated Inhibition of Oncogenic PLK1 in B-Cell Acute Lymphoblastic Leukemia.

In B-cell acute lymphoblastic leukemia (B-ALL), activation of Notch signaling leads to cell-cycle arrest and apoptosis. We aimed to harness knowledge acquired by understanding a mechanism of Notch-induced cell death to elucidate a therapeutically viable target in B-ALL. To this end, we identified that Notch activation suppresses Polo-like kinase 1 (PLK1) in a B-ALL-specific manner. We identified that PLK1 is expressed in all subsets of B-ALL and is highest in Philadelphia-like (Ph-like) ALL, a high-risk subtype of disease. We biochemically delineated a mechanism of Notch-induced PLK1 downregulation that elucidated stark regulation of p53 in this setting. Our findings identified a novel posttranslational cascade initiated by Notch in which CHFR was activated via PARP1-mediated PARylation, resulting in ubiquitination and degradation of PLK1. This led to hypophosphorylation of MDM2Ser260, culminating in p53 stabilization and upregulation of BAX. shRNA knockdown or pharmacologic inhibition of PLK1 using BI2536 or BI6727 (volasertib) in B-ALL cell lines and patient samples led to p53 stabilization and cell death. These effects were seen in primary human B-ALL samples in vitro and in patient-derived xenograft models in vivo These results highlight PLK1 as a viable therapeutic target in B-ALL. Efficacy of clinically relevant PLK1 inhibitors in B-ALL patient-derived xenograft mouse models suggests that use of these agents may be tailored as an additional therapeutic strategy in future clinical studies.