Exploiting a key transcriptional dependency: ZMYND8 and IRF8 in AML.

In this issue of Molecular Cell, Cao et al. (2021) report that AML cells are specifically addicted to an IRF8-MEF2D gene expression network. Furthermore, they identify a chromatin reader, ZMYND8, as the upstream regulator of the IRF8-MEF2D program whose activity is critical for AML cell survival.

CENP-C-targeted PLK-1 regulates kinetochore function in C. elegans embryos.

Polo-like kinase 1 (PLK-1) is present in centrosomes, the nuclear envelope, and kinetochores and plays a significant role in meiosis and mitosis. PLK-1 depletion or inhibition has severe consequences for spindle assembly, spindle assembly checkpoint (SAC) activation, chromosome segregation, and cytokinesis. BUB-1 targets PLK-1 to the outer kinetochore and, in mammals, the inner kinetochore PLK1 targeting is mediated by the constitutive centromere associated network (CCAN). BUB1-targeted PLK-1 plays a key role in SAC activation and a SAC-independent role through targeting CDC-20. In contrast, whether there is a specific, non-redundant role for inner kinetochore targeted PLK-1 is unknown. Here, we used the C. elegans embryo to study the role of inner kinetochore PLK-1. We found that CENP-C, the sole CCAN component in C. elegans and other species, targets PLK-1 to the inner kinetochore during prometaphase and metaphase. Disruption of the CENP-C/PLK-1 interaction leads to an imbalance in kinetochore components and a defect in chromosome congression, without affecting CDC-20 recruitment. These findings indicate that PLK-1 kinetochore recruitment by CENP-C has at least partially distinct functions than outer kinetochore PLK-1, providing a platform for a better understanding of the different roles played by PLK-1 during mitosis.

The DNA dioxygenase Tet1 regulates H3K27 modification and embryonic stem cell biology independent of its catalytic activity.

Tet enzymes (Tet1/2/3) oxidize 5-methylcytosine to promote DNA demethylation and partner with chromatin modifiers to regulate gene expression. Tet1 is highly expressed in embryonic stem cells (ESCs), but its enzymatic and non-enzymatic roles in gene regulation are not dissected. We have generated Tet1 catalytically inactive (Tet1m/m) and knockout (Tet1-/-) ESCs and mice to study these functions. Loss of Tet1, but not loss of its catalytic activity, caused aberrant upregulation of bivalent (H3K4me3+; H3K27me3+) developmental genes, leading to defects in differentiation. Wild-type and catalytic-mutant Tet1 occupied similar genomic loci which overlapped with H3K27 tri-methyltransferase PRC2 and the deacetylase complex Sin3a at promoters of bivalent genes and with the helicase Chd4 at active genes. Loss of Tet1, but not loss of its catalytic activity, impaired enrichment of PRC2 and Sin3a at bivalent promoters leading to reduced H3K27 trimethylation and deacetylation, respectively, in absence of any changes in DNA methylation. Tet1-/-, but not Tet1m/m, embryos expressed higher levels of Gata6 and were developmentally delayed. Thus, the critical functions of Tet1 in ESCs and early development are mediated through its non-catalytic roles in regulating H3K27 modifications to silence developmental genes, and are more important than its catalytic functions in DNA demethylation.

From the United States to the United Kingdom: Hope, Blended Family Social Support, and Computer-Mediated Communication Amidst Chronic Illness.

This Defining Moments essay contributes to health communication scholarship regarding the role of religion/spirituality as a coping strategy during severe illness and the realities of providing social support in blended families. It also invites readers to reconsider the capacities and constraints of utilizing technology-mediated communication to provide support within close familial relationships. Finally, this narrative acknowledges the messiness of providing support in the evolving realities of family life and the complexity of "being there" for people we love when we cannot physically be present.

Disparity in peripheral and renal B-cell depletion with rituximab in systemic lupus erythematosus: an opportunity for obinutuzumab?

OBJECTIVES: To investigate key factors that may contribute to the variability of rituximab-mediated peripheral and renal B cell depletion (BCD) in SLE. METHODS: We analysed: (i) CD19+ B cell counts in patients with SLE before and 1, 2, 3 and 6 months after treatment with rituximab, comparing them with RA patients; (ii) the presence of B cells in renal biopsies after rituximab therapy; (iii) whether the duration of BCD correlated with patient demographics and B cell expression of CD20 and FcγRIIb; and (iv) the effect of B cell activation factor (BAFF) on the efficiency of rituximab and obinutuzumab at inducing BCD in whole blood assays, in vitro. RESULTS: In SLE (n = 71), the duration of BCD was shorter compared with RA (n = 27). B cells were detectable in renal biopsy samples (n = 6) after treatment with rituximab in all patients with poor response while peripheral blood B cells remained low or undetectable in the same patients. There were no significant relationships between peripheral BCD and patient age, disease duration, serum C3 levels or the level of expression of B cell surface proteins CD20 and FcγRIIb. Obinutuzumab was more efficient than rituximab at inducing BCD in whole blood assays, regardless of excess BAFF. CONCLUSIONS: BCD in SLE is less efficient than in RA. Renal B cell presence following rituximab treatment was associated with poor outcomes. No significant relationships between any measured B cell related, clinical or laboratory parameters and the efficiency of BCD by rituximab was found. Obinutuzumab was superior to rituximab at inducing BCD.

Runx1 promotes murine erythroid progenitor proliferation and inhibits differentiation by preventing Pu.1 downregulation.

Pu.1 is an ETS family transcription factor (TF) that plays critical roles in erythroid progenitors by promoting proliferation and blocking terminal differentiation. However, the mechanisms controlling expression and down-regulation of Pu.1 during early erythropoiesis have not been defined. In this study, we identify the actions of Runx1 and Pu.1 itself at the Pu.1 gene Upstream Regulatory Element (URE) as major regulators of Pu.1 expression in Burst-Forming Unit erythrocytes (BFUe). During early erythropoiesis, Runx1 and Pu.1 levels decline, and chromatin accessibility at the URE is lost. Ectopic expression of Runx1 or Pu.1, both of which bind the URE, prevents Pu.1 down-regulation and blocks terminal erythroid differentiation, resulting in extensive ex vivo proliferation and immortalization of erythroid progenitors. Ectopic expression of Runx1 in BFUe lacking a URE fails to block terminal erythroid differentiation. Thus, Runx1, acting at the URE, and Pu.1 itself directly regulate Pu.1 levels in erythroid cells, and loss of both factors is critical for Pu.1 down-regulation during terminal differentiation. The molecular mechanism of URE inactivation in erythroid cells through loss of TF binding represents a distinct pattern of Pu.1 regulation from those described in other hematopoietic cell types such as T cells which down-regulate Pu.1 through active repression. The importance of down-regulation of Runx1 and Pu.1 in erythropoiesis is further supported by genome-wide analyses showing that their DNA-binding motifs are highly overrepresented in regions that lose chromatin accessibility during early erythroid development.

Flt3 inhibitor AC220 is a potent therapy in a mouse model of myeloproliferative disease driven by enhanced wild-type Flt3 signaling.

High levels of expression of wild-type Flt3 characterize many hematopoietic proliferative diseases and neoplasms, providing a potential therapeutic target. Using the c-Cbl RING finger mutant mouse as a model of a myeloproliferative disease (MPD) driven by wild-type Flt3, in the present study, we show that treatment with the Flt3 kinase inhibitor AC220 blocks MPD development by targeting Flt3(+) multipotent progenitors (MPPs). We found that daily administration of AC220 caused a marked reduction in Flt3 expression, induction of quiescence, and a significant loss of MPPs within 4 days. Unexpectedly, a robust Flt3 ligand-associated proliferative recovery response soon followed, preventing further loss of MPPs. However, continued AC220 treatment limited MPP recovery and maintained reduced, steady-state levels of cycling MPPs that express low levels of Flt3. Therefore, a finely tuned balance between the opposing forces of AC220 and Flt3 ligand production was established; whereas the Flt3 ligand blunted the inhibitory effects of AC220, the disease was held in remission for as long as therapy was continued. The net effect is a potent therapy indicating that patients with c-Cbl mutations, or those with similarly enhanced Flt3 signaling, may respond well to AC220 even after the induction of high levels of Flt3 ligand.

Pharmacological restriction of genomic binding sites redirects PU.1 pioneer transcription factor activity.

Transcription factor (TF) DNA-binding dynamics govern cell fate and identity. However, our ability to pharmacologically control TF localization is limited. Here we leverage chemically driven binding site restriction leading to robust and DNA-sequence-specific redistribution of PU.1, a pioneer TF pertinent to many hematopoietic malignancies. Through an innovative technique, 'CLICK-on-CUT&Tag', we characterize the hierarchy of de novo PU.1 motifs, predicting occupancy in the PU.1 cistrome under binding site restriction. Temporal and single-molecule studies of binding site restriction uncover the pioneering dynamics of native PU.1 and identify the paradoxical activation of an alternate target gene set driven by PU.1 localization to second-tier binding sites. These transcriptional changes were corroborated by genetic blockade and site-specific reporter assays. Binding site restriction and subsequent PU.1 network rewiring causes primary human leukemia cells to differentiate. In summary, pharmacologically induced TF redistribution can be harnessed to govern TF localization, actuate alternate gene networks and direct cell fate.

BUB-1 and CENP-C recruit PLK-1 to control chromosome alignment and segregation during meiosis I in C. elegans oocytes.

Phosphorylation is a key post-translational modification that is utilised in many biological processes for the rapid and reversible regulation of protein localisation and activity. Polo-like kinase 1 (PLK-1) is essential for both mitotic and meiotic cell divisions, with key functions being conserved in eukaryotes. The roles and regulation of PLK-1 during mitosis have been well characterised. However, the discrete roles and regulation of PLK-1 during meiosis have remained obscure. Here, we used Caenorhabditis elegans oocytes to show that PLK-1 plays distinct roles in meiotic spindle assembly and/or stability, chromosome alignment and segregation, and polar body extrusion during meiosis I. Furthermore, by a combination of live imaging and biochemical analysis we identified the chromosomal recruitment mechanisms of PLK-1 during C. elegans oocyte meiosis. The spindle assembly checkpoint kinase BUB-1 directly recruits PLK-1 to the kinetochore and midbivalent while the chromosome arm population of PLK-1 depends on a direct interaction with the centromeric-associated protein CENP-CHCP-4. We found that perturbing both BUB-1 and CENP-CHCP-4 recruitment of PLK-1 leads to severe meiotic defects, resulting in highly aneuploid oocytes. Overall, our results shed light on the roles played by PLK-1 during oocyte meiosis and provide a mechanistic understanding of PLK-1 targeting to meiotic chromosomes.

DNA selection by the master transcription factor PU.1.

The master transcriptional regulator PU.1/Spi-1 engages DNA sites with affinities spanning multiple orders of magnitude. To elucidate this remarkable plasticity, we have characterized 22 high-resolution co-crystallographic PU.1/DNA complexes across the addressable affinity range in myeloid gene transactivation. Over a purine-rich core (such as 5'-GGAA-3') flanked by variable sequences, affinity is negotiated by direct readout on the 5' flank via a critical glutamine (Q226) sidechain and by indirect readout on the 3' flank by sequence-dependent helical flexibility. Direct readout by Q226 dynamically specifies PU.1's characteristic preference for purines and explains the pathogenic mutation Q226E in Waldenström macroglobulinemia. The structures also reveal how disruption of Q226 mediates strand-specific inhibition by DNA methylation and the recognition of non-canonical sites, including the authentic binding sequence at the CD11b promoter. A re-synthesis of phylogenetic and structural data on the ETS family, considering the centrality of Q226 in PU.1, unifies the model of DNA selection by ETS proteins.

Influence of influenza A infection on capsaicin-induced responses in murine airways.

The principal aim of the study was to determine the influence of influenza A virus infection on capsaicin-induced relaxation responses in mouse isolated tracheal segments and clarify the underlying mechanisms. Anesthetized mice were intranasally inoculated with influenza A/PR-8/34 virus (VIRUS) or vehicle (SHAM), and 4 days later tracheal segments were harvested for isometric tension recording and biochemical and histologic analyses. Capsaicin induced dose-dependent relaxation responses in carbachol-contracted SHAM trachea (e.g., 10 µM capsaicin produced 66 ± 4% relaxation; n = 11), which were significantly inhibited by capsazepine [transient receptor potential vanilloid type 1 (TRPV1) antagonist], (2S,3S)-3-{[3,5-bis(trifluoromethyl)phenyl]methoxy}-2-phenylpiperidine hydrochloride (L-733,060) [neurokinin 1 (NK1) receptor antagonist], indomethacin [cyclooxygenase (COX) inhibitor], and the combination of 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH6809) and 7-[5α-([1S,1α(Z)-biphenyl]-4-ylmethoxy)-2ß-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid, calcium salt, hydrate (AH23848) [E-prostanoid (EP)2 and EP4 receptor antagonists, respectively], indicating that capsaicin-induced relaxation involved the TRPV1-mediated release of substance P (SP), activation of epithelial NK1 receptors, and production of COX products capable of activating relaxant EP2/EP4 receptors. Consistent with this postulate, capsaicin-induced relaxation was associated with the significant release of SP and prostaglandin E2 (PGE2) from mouse tracheal segments. As expected, influenza A virus infection was associated with widespread disruption of the tracheal epithelium. Tracheal segments from VIRUS mice responded weakly to capsaicin (7 ± 3% relaxation) and were 25-fold less responsive to SP than tracheas from SHAM mice. In contrast, relaxation responses to exogenous PGE2 and the ß-adrenoceptor agonist isoprenaline were not inhibited in VIRUS trachea. Virus infection was associated with impaired capsaicin-induced release of PGE2, but the release of SP was not affected. In summary, influenza A virus infection profoundly inhibits capsaicin- and SP-induced relaxation responses, most likely by inhibiting the production of PGE2.

MDMX acts as a pervasive preleukemic-to-acute myeloid leukemia transition mechanism.

MDMX is overexpressed in the vast majority of patients with acute myeloid leukemia (AML). We report that MDMX overexpression increases preleukemic stem cell (pre-LSC) number and competitive advantage. Utilizing five newly generated murine models, we found that MDMX overexpression triggers progression of multiple chronic/asymptomatic preleukemic conditions to overt AML. Transcriptomic and proteomic studies revealed that MDMX overexpression exerts this function, unexpectedly, through activation of Wnt/ß-Catenin signaling in pre-LSCs. Mechanistically, MDMX binds CK1α and leads to accumulation of ß-Catenin in a p53-independent manner. Wnt/ß-Catenin inhibitors reverse MDMX-induced pre-LSC properties, and synergize with MDMX-p53 inhibitors. Wnt/ß-Catenin signaling correlates with MDMX expression in patients with preleukemic myelodysplastic syndromes and is associated with increased risk of progression to AML. Our work identifies MDMX overexpression as a pervasive preleukemic-to-AML transition mechanism in different genetically driven disease subtypes, and reveals Wnt/ß-Catenin as a non-canonical MDMX-driven pathway with therapeutic potential for progression prevention and cancer interception.

Chromosome segregation during female meiosis in C. elegans: A tale of pushing and pulling.

The role of the kinetochore during meiotic chromosome segregation in C. elegans oocytes has been a matter of controversy. Danlasky et al. (2020. J. Cell. Biol.https://doi.org/10.1083/jcb.202005179) show that kinetochore proteins KNL-1 and KNL-3 are required for early stages of anaphase during female meiosis, suggesting a new kinetochore-based model of chromosome segregation.

BUB-1 targets PP2A:B56 to regulate chromosome congression during meiosis I in C. elegans oocytes.

Protein Phosphatase 2A (PP2A) is a heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits. PP2A complexes with B56 subunits are targeted by Shugoshin and BUBR1 to protect centromeric cohesion and stabilise kinetochore-microtubule attachments in yeast and mouse meiosis. In Caenorhabditis elegans, the closest BUBR1 orthologue lacks the B56-interaction domain and Shugoshin is not required for meiotic segregation. Therefore, the role of PP2A in C. elegans female meiosis is unknown. We report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. BUB-1 is the main chromosome-targeting factor for B56 subunits during prometaphase I. BUB-1 recruits PP2A:B56 to the chromosomes via a newly identified LxxIxE motif in a phosphorylation-dependent manner, and this recruitment is important for proper chromosome congression. Our results highlight a novel mechanism for B56 recruitment, essential for recruiting a pool of PP2A involved in chromosome congression during meiosis I.

Bony Patchwork: Mosaic Patterns of Evolution in the Skull of Electric Fishes (Apteronotidae: Gymnotiformes).

Mosaic evolution refers to the pattern whereby different organismal traits exhibit differential rates of evolution typically due to reduced levels of trait covariation through deep time (i.e., modularity). These differences in rates can be attributed to variation in responses to selective pressures between individual traits. Differential responses to selective pressures also have the potential to facilitate functional specialization, allowing certain traits to track environmental stimuli more closely than others. The teleost skull is a multifunctional structure comprising a complex network of bones and thus an excellent system for which to study mosaic evolution. Here we construct an ultrametric phylogeny for a clade of Neotropical electric fishes (Apteronotidae: Gymnotiformes) and use three-dimensional geometric morphometrics to investigate patterns of mosaic evolution in the skull and jaws. We find strong support for a developmental, three-module hypothesis that consists of the face, braincase, and mandible, and we find that the mandible has evolved four times faster than its neighboring modules. We hypothesize that the functional specialization of the mandible in this group of fishes has allowed it to outpace the face and braincase and evolve in a more decoupled manner. We also hypothesize that this pattern of mosaicism may be widespread across other clades of teleost fishes.

IL1RAP potentiates multiple oncogenic signaling pathways in AML.

The surface molecule interleukin-1 receptor accessory protein (IL1RAP) is consistently overexpressed across multiple genetic subtypes of acute myeloid leukemia (AML) and other myeloid malignancies, including at the stem cell level, and is emerging as a novel therapeutic target. However, the cell-intrinsic functions of IL1RAP in AML cells are largely unknown. Here, we show that targeting of IL1RAP via RNA interference, genetic deletion, or antibodies inhibits AML pathogenesis in vitro and in vivo, without perturbing healthy hematopoietic function or viability. Furthermore, we found that the role of IL1RAP is not restricted to the IL-1 receptor pathway, but that IL1RAP physically interacts with and mediates signaling and pro-proliferative effects through FLT3 and c-KIT, two receptor tyrosine kinases with known key roles in AML pathogenesis. Our study provides a new mechanistic basis for the efficacy of IL1RAP targeting in AML and reveals a novel role for this protein in the pathogenesis of the disease.

Hitting the snooze button: Inducing quiescence with the FLT3 inhibitor quizartinib protects hematopoietic progenitors from chemotherapy.

Myelosuppression is one of the most severe and limiting side effects of chemotherapy. Our recent work outlines a strategy to prevent chemotherapy-induced myelosuppression by administering a priming dose of the FMS-Like Tyrosine kinase 3 (FLT3) inhibitor quizartinib. Furthermore, by administering sequential quizartinib primed injections of fluorouracil (5-FU), we demonstrated a novel and effective strategy to eliminate disease in two mouse models of quizartinib resistant acute myeloid leukemia (AML).

Preventing chemotherapy-induced myelosuppression by repurposing the FLT3 inhibitor quizartinib.

We describe an approach to inhibit chemotherapy-induced myelosuppression. We found that short-term exposure of mice to the FLT3 inhibitor quizartinib induced the transient quiescence of multipotent progenitors (MPPs). This property of quizartinib conferred marked protection to MPPs in mice receiving fluorouracil or gemcitabine. The protection resulted in the rapid recovery of bone marrow and blood cellularity, thus preventing otherwise lethal myelosuppression. A treatment strategy involving quizartinib priming that protected wild-type bone marrow progenitors, but not leukemic cells, from fluorouracil provided a more effective treatment than conventional induction therapy in mouse models of acute myeloid leukemia. This strategy has the potential to be extended for use in other cancers where FLT3 inhibition does not adversely affect the effectiveness of chemotherapy. Thus, the addition of quizartinib to cancer treatment regimens could markedly improve cancer patient survival and quality of life.

Dasatinib promotes the activation of quiescent hematopoietic stem cells in mice.

Dasatinib is an orally available broad-spectrum tyrosine kinase inhibitor that is widely used to treat chronic myeloid leukemia. It is also in clinical trials for the treatment of other malignancies, including solid tumors. Despite its wide use, little is known of its effects on normal hematopoietic stem and progenitor cells. Here, we study wild-type mice dosed with dasatinib and find that it causes the transient induction of proliferation of quiescent hematopoietic stem cells (HSCs). This finding was unexpected given the ability of dasatinib to inhibit c-Kit signaling and promote cell cycle arrest in many cell types. The transient induction of HSC proliferation in dasatinib-dosed mice coincided with a marked induction in the expression of Sca-1 and phospho-S6. Also evident at this time was a rapid but transient loss of lineage-committed hematopoietic progenitors that express high levels of c-Kit and the induction of stem cell factor in the serum. These findings suggest that activation of quiescent HSCs is part of a rapid rescue response that restores hematopoietic progenitors to pretreatment levels. This restoration coincides with HSCs returning to quiescence, and the expression of Sca-1 and phospho-S6 reverting to pre-treatment levels, even though dasatinib dosing is maintained. These data suggest that equilibrium is reached between the opposing forces of dasatinib and hematopoietic growth factors. The transient induction of HSC proliferation provided a window of opportunity whereby these cells became sensitive to killing by the cytotoxic drug 5-fluorouracil.