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Acute leukemia is a heterogeneous disorder of hematologic malignancies composed primarily of hematopoietic precursors that have acquired unregulated self‐renewal and proliferation. Hematology classification systems typically divide these neoplasms into lymphoid (B‐ or T‐) and myeloid‐lineage subtypes, with therapy dependent upon this distinction. Infrequently, certain acute leukemias may undergo a complete lineage switch at relapse, subsequently complicating the diagnosis and treatment of these recurrent diseases. Transformation from B‐lineage to myeloid lineage is the most common switch observed, and is frequently associated with a balanced 11q23 translocation, involving KMT2A. The mechanisms involved in the lineage‐switch are unclear, but modern therapies targeting the B‐cell‐specific marker, CD19, have proven to promote this conversion as one means of treatment escape. Broadly speaking, therapy‐mediated selection of alternate lineage‐committed subclones derived from the same initial pluripotent progenitors, clonal evolution and reprogramming of lineage‐committed blasts, and de novo clonally unrelated leukemias may account for the clinical impression of lineage switched acute leukemia during treatment. This review will explore the phenomenon and potential mechanisms of lineage transformation during the treatment of acute leukemia. [ABSTRACT FROM AUTHOR]

Fluorescence anisotropy capillary electrophoresis (FACE) and affinity probe capillary electrophoresis (APCE) with laser-induced fluorescence detection were evaluated for analysis of peptide--protein interactions with rapid binding kinetics. The Src homology 2 domain of protein SH2-B[beta] (SH2-B[beta] (525-670)) and a tyrosine-phosphorylated peptide corresponding to the binding sequence of JAK2 were used as a model system. For peptide labeled with fluorescein, the [K.sub.d] = 82 [+ or -] 7 nM as measured by fluorescence anisotropy (FA). APCE assays had a limit of detection (LOD) of 100 nM or 12 amol injected for SH2-B[beta] (525-670). The separation time of 4 s, achieved using an electric field of 2860 V/cm on 7-cm-long capillaries, was on the same time scale as complex dissociation allowing [K.sub.d] (101 [+ or -] 12 nM in good agreement with FA measurements) and dissociation rate ([k.sub.off] = 0.95 [+ or -] 0.02 [s.sup.-1] corresponding to a half-life of 0.73 s) to be determined. This measurement represents a 30-fold higher rate of complex dissociation than what had previously been measurable by nonequilibrium CE analysis of equilibrium mixtures. Using FACE, the protein was detected with an LOD of 300 nM or 7.5 fmol injected. FACE was not used for determining [K.sub.d] or [k.sub.off]; however, this method provided better separation resolution for multiple forms of the protein than APCE. Both methods were found suitable for analysis of cell lysate. These results demonstrate that FACE and APCE may be useful complements to existing techniques for exploring binding interactions with rapid kinetics.

Next generation sequencing has uncovered several genes with associated mutations in hematologic malignancies that can serve as potential biomarkers of disease. Keeping abreast of these genes is therefore of paramount importance in the field of hematology. This review focuses on PHF6 , a highly conserved epigenetic transcriptional regulator that is important for neurodevelopment and hematopoiesis. PHF 6 serves as a tumor suppressor protein, with PHF6 mutations and deletions often implicated in the development of T-lymphoblastic leukemia and less frequently in acute myeloid leukemia and other myeloid neoplasms. PHF6 inactivation appears to be an early event in T-lymphoblastic leukemogenesis, requiring cooperating events, including NOTCH1 mutations or overexpression of TLX1 and TLX3 for full disease development. In contrast, PHF6 mutations tend to occur later in myeloid malignancies, are frequently accompanied by RUNX1 mutations, and are often associated with disease progression. Moreover, PHF 6 appears to play a role in lineage plasticity within hematopoietic malignancies, with PHF6 mutations commonly present in mixed phenotype acute leukemias with a predilection for T-lineage marker expression. Due to conflicting data, the prognostic significance of PHF6 mutations remains unclear, with a subset of studies showing no significant difference in outcomes compared to malignancies with wild-type PHF6 , and other studies showing inferior outcomes in certain patients with mutated PHF6. Future studies are necessary to elucidate the role PHF6 plays in development of T-lymphoblastic leukemia, progression of myeloid malignancies, and its overall prognostic significance in hematopoietic neoplasms. [ABSTRACT FROM AUTHOR]

Background: Fine‐needle aspiration with flow cytometry (FNA‐FC) is routinely used in the evaluation of lymph nodes suspicious for lymphoma, yet data comparing immunophenotype distributions and outliers in benign lymph nodes sampled by fine‐needle aspiration (FNA) versus excision are lacking. Methods: Flow cytometry data from 289 benign lymph node FNA cases were assessed for the overall antigen distribution, with a focus on outliers relevant to the diagnosis of lymphoma. Distributions and outlier proportions were compared with those of a separate cohort of 298 excisional biopsies. Results: Compared with excisional biopsies, FNA specimens overrepresented CD3+ events (72% vs 63%), underrepresented CD19+ events (22% vs 29%), and had 25% fewer large cell–gated events. Normalized antigen distributions in FNA were equivalent to those in excisional biopsy. Twenty‐three percent of FNA‐FC cases exhibited an outlier, including a skewed kappa:lambda light‐chain ratio, increased CD5+ or CD10+ B‐cell events, a skewed CD4:CD8 ratio, and increased CD7 loss on T cells, with no significant differences in frequency or type in comparison with excisional specimens. Outliers for the light‐chain ratio and T‐cell antigens were enriched among older patients and included patients with a variety of autoimmune/rheumatologic conditions. Conclusions: Benign lymph node FNA yields flow immunophenotypes remarkably similar to those from excisional biopsies. Outlier flow immunophenotypes are identified in benign lymph nodes sampled by FNA at a frequency similar to that with excisional biopsies. Older patients, who have a higher baseline risk of lymphoma, are more likely to exhibit lymphoma‐mimicking outliers such as a light‐chain predominance on B cells and skewed CD4:CD8 ratios or increased CD7 loss on T cells, and they warrant additional diagnostic caution. Fine‐needle aspiration–based flow cytometry of lymph nodes is comparable to excisional biopsy and exhibits similar outliers. The reference ranges provided in this study can aid pathologists and contribute in the long term to computational flow analysis and biomedical research. [ABSTRACT FROM AUTHOR]

Summary Acute lymphoblastic leukemia (ALL) is associated with significant morbidity and mortality, necessitating further improvements in diagnosis and therapy. Targeted therapies directed against chromatin regulators are emerging as promising approaches in preclinical studies and early clinical trials. Here, we demonstrate an oncogenic role for the protein lysine methyltransferase SETDB2 in leukemia pathogenesis. It is overexpressed in pre-BCR + ALL and required for their maintenance in vitro and in vivo . SETDB2 expression is maintained as a direct target gene of the chimeric transcription factor E2A-PBX1 in a subset of ALL and suppresses expression of the cell-cycle inhibitor CDKN2C through histone H3K9 tri-methylation, thus establishing an oncogenic pathway subordinate to E2A-PBX1 that silences a major tumor suppressor in ALL. In contrast, SETDB2 was relatively dispensable for normal hematopoietic stem and progenitor cell proliferation. SETDB2 knockdown enhances sensitivity to kinase and chromatin inhibitors, providing a mechanistic rationale for targeting SETDB2 therapeutically in ALL. [ABSTRACT FROM AUTHOR]

Objectives: We retrospectively tested the prognostic and diagnostic significance of CD271+ mesenchymal stromal cell (MSC) density in cytopenic patients who underwent bone marrow biopsy to evaluate for myelodysplastic syndromes (MDS). Methods: CD271+ MSC density was quantitated by automated image analysis of tissue microarray cores in 125 cytopenic patients: 40 lower grade MDS (<5% marrow blasts), 24 higher grade MDS, and 61 benign. Results: CD271+ MSC density was increased in higher grade MDS compared with benign (P = .006) and lower grade MDS (P = .02). CD271+ MSC density was predictive of survival among patients with MDS independent of Revised International Prognostic Scoring System (IPSS-R), history of transfusion, therapy-related MDS, and fibrosis (hazard ratio, 3.4; P < .001). Among low or intermediate IPSS-R patients, median survival was significantly shorter in the high CD271+ MSC density group (47 vs 18 months, P < .02). Conclusions: High CD271+ MSC density is characteristic of higher grade MDS and is associated with poor risk independent of known prognostic factors. [ABSTRACT FROM AUTHOR]

The tyrosine kinase Janus kinase 2 (JAK2) binds to the majority of the known members of the cytokine family of receptors. Ligand-receptor binding leads to activation of the associated JAK2 molecules, resulting in rapid autophosphorylation of multiple tyrosines within JAK2. Phosphotyrosines can then serve as docking sites for downstream JAK2 signaling molecules. Despite the importance of these phosphotyrosines in JAK2 function, only a few sites and binding partners have been identified. Using two-dimensional phosphopeptide mapping and a phosphospecific antibody, we identified tyrosine 813 as a site of JAK2 autophosphorylation of overexpressed JAK2 and endogenous JAK2 activated by growth hormone. Tyrosine 813 is contained within a YXXL sequence motif associated with several other identified JAK2 phosphorylation sites. We show that phosphorylation of tyrosine 813 is required for the SH2 domain-containing adapter protein SH2-Bβ to bind JAK2 and to enhance the activity of JAK2 and STAT5B. The homologous tyrosine in JAK3, tyrosine 785, is autophosphorylated in response to interleukin-2 stimulation and is required for SH2-Bβ to bind JAK3. Taken together these data strongly suggest that tyrosine 813 is a site of autophosphorylation in JAK2 and is the SH2-Bβ-binding site within JAK2 that is required for SH2-Bβ to enhance activation of JAK2. [ABSTRACT FROM AUTHOR]

The tyrosine kinase Janus kinase 2 (JAK2) transduces signaling for the majority of known cytokine receptor family members and is constitutively activated in some cancers. Here we examine the mechanisms by which the adapter proteins SH2-Bß and APS regulate the activity of JAK2. We show that like SH2-Bß, APS binds JAK2 at multiple sites and that binding to phosphotyrosine 813 is essential for APS to increase active JAK2 and to be phosphorylated by JAK2. Binding of APS to a phosphotyrosine 813-independent site inhibits JAK2. Both APS and SH2-Bß increase JAK2 activity independent of their N-terminal dimerization domains. SH2-Bß-induced increases in JAK2 dimerization require only the SH2 domain and only one SH2-Bß to be bound to a JAK2 dimer. JAK2 mutations and truncations revealed that amino acids 809 to 811 in JAK2 are a critical component of a larger regulatory region within JAK2, most likely including amino acids within the JAK homology 1 (JH1) and JH2 domains and possibly the FERM domain. Together, our data suggest that SH2-Bß and APS do not activate JAK2 as a consequence of their own dimerization, recruitment of an activator of JAK2, or direct competition with a JAK2 inhibitor for binding to JAK2. Rather, they most likely induce or stabilize an active conformation of JAK2. [ABSTRACT FROM AUTHOR]

Fluorescence anisotropy capillary electrophoresis (FACE) and affinity probe capillary electrophoresis (APCE) with laser-induced fluorescence detection were evaluated for analysis of peptide-protein interactions with rapid bind- big kinetics. The Src homology 2 domain of protein SH2- Bβ (SH2-Bβ (525-670)) and a tyrosine-phosphorylated peptide corresponding to the binding sequence of JAK2 were used as a model system. For peptide labeled with fluorescein, the Kd = 82 ± 7 mM as measured by fluorescence anisotropy (FA). APCE assays had a limit of detection (LOD) of 100 nM or 12 amol injected for SH2- Bβ (525-670). The separation time of 4 s, achieved using an electric field of 2860 V/cm on 7-cm-long capillaries, was on the same time scale as complex dissociation allowing Kd (101 ± 12 nM in good agreement with FA measurements) and dissociation rate (koff = 0.95 ± 0.02 s-1 corresponding to a half-life of 0.73 s) to be determined. This measurement represents a 30-fold higher rate of complex dissociation than what had previously been measurable by nonequilibrium CE analysis of equilibrium mixtures. Using FACE, the protein was detected with an LOD of 300 nM or 7.5 fmol injected. FACE was not used for determining Kd or koff; however, this method provided better separation resolution for multiple forms of the protein than APCE. Both methods were found suitable for analysis of cell lysate. These results demonstrate that FACE and APCE may be useful complements to existing techniques for exploring binding interactions with rapid kinetics. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is a type of cancer caused by aggressive neoplastic proliferations of immature myeloid cells that is fatal if untreated. AML accounts for 1.0% of all new cancer cases in the United States, with a 5-year relative survival rate of 30.5%. Once defined primarily morphologically, advances in next generational sequencing have expanded the role of molecular genetics in categorizing the disease. As such, both the World Health Organization Classification of Haematopoietic Neoplasms and The International Consensus Classification System now define a variety of AML subsets based on mutations in driver genes such as NPM1, CEBPA, TP53, ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, and ZRSR2. This article provides an overview of some of the genetic mutations associated with AML and compares how the new classification systems incorporate molecular genetics into the definition of AML., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023. Published by Elsevier Inc.)

There is limited understanding of how signaling pathways are altered by oncogenic fusion transcription factors that drive leukemogenesis. To address this, we interrogated activated signaling pathways in a comparative analysis of mouse and human leukemias expressing the fusion protein E2A-PBX1, which is present in 5%-7% of pediatric and 50% of pre-B-cell receptor (preBCR + ) acute lymphocytic leukemia (ALL). In this study, we describe remodeling of signaling networks by E2A-PBX1 in pre-B-ALL, which results in hyperactivation of the key oncogenic effector enzyme PLCγ2. Depletion of PLCγ2 reduced proliferation of mouse and human ALLs, including E2A-PBX1 leukemias, and increased disease-free survival after secondary transplantation. Mechanistically, E2A-PBX1 bound promoter regulatory regions and activated the transcription of its key target genes ZAP70, SYK, and LCK, which encode kinases upstream of PLCγ2. Depletion of the respective upstream kinases decreased cell proliferation and phosphorylated levels of PLCγ2 (pPLCγ2). Pairwise silencing of ZAP70, SYK, or LCK showed additive effects on cell growth inhibition, providing a rationale for combination therapy with inhibitors of these kinases. Accordingly, inhibitors such as the SRC family kinase (SFK) inhibitor dasatinib reduced pPLCγ2 and inhibited proliferation of human and mouse preBCR + /E2A-PBX1 + leukemias in vitro and in vivo Furthermore, combining small-molecule inhibition of SYK, LCK, and SFK showed synergistic interactions and preclinical efficacy in the same setting. Our results show how the oncogenic fusion protein E2A-PBX1 perturbs signaling pathways upstream of PLCγ2 and renders leukemias amenable to targeted therapeutic inhibition. Cancer Res; 76(23); 6937-49. ©2016 AACR., (©2016 American Association for Cancer Research.)

Fluorescence anisotropy capillary electrophoresis (FACE) and affinity probe capillary electrophoresis (APCE) with laser-induced fluorescence detection were evaluated for analysis of peptide-protein interactions with rapid binding kinetics. The Src homology 2 domain of protein SH2-Bbeta (SH2-Bbeta (525-670)) and a tyrosine-phosphorylated peptide corresponding to the binding sequence of JAK2 were used as a model system. For peptide labeled with fluorescein, the K(d) = 82 +/- 7 nM as measured by fluorescence anisotropy (FA). APCE assays had a limit of detection (LOD) of 100 nM or 12 amol injected for SH2-Bbeta (525-670). The separation time of 4 s, achieved using an electric field of 2860 V/cm on 7-cm-long capillaries, was on the same time scale as complex dissociation allowing K(d) (101 +/- 12 nM in good agreement with FA measurements) and dissociation rate (k(off) = 0.95 +/- 0.02 s(-)(1) corresponding to a half-life of 0.73 s) to be determined. This measurement represents a 30-fold higher rate of complex dissociation than what had previously been measurable by nonequilibrium CE analysis of equilibrium mixtures. Using FACE, the protein was detected with an LOD of 300 nM or 7.5 fmol injected. FACE was not used for determining K(d) or k(off); however, this method provided better separation resolution for multiple forms of the protein than APCE. Both methods were found suitable for analysis of cell lysate. These results demonstrate that FACE and APCE may be useful complements to existing techniques for exploring binding interactions with rapid kinetics.