The extracellular matrix microtopography drives critical changes in cellular motility and Rho A activity in colon cancer cells

We have shown that the microtopography (mT) underlying colon cancer changes as a tumor de-differentiates. We distinguish the well-differentiated mT based on the increasing number of "pits" and poorly differentiated mT on the basis of increasing number of "posts." We investigated Rho A as a mechanosensing protein using mT features derived from those observed in the ECM of colon cancer. We evaluated Rho A activity in less-tumorogenic (Caco-2 E) and more tumorigenic (SW620) colon cancer cell-lines on microfabricated pits and posts at 2.5 μm diameter and 200 nm depth/height. In Caco-2 E cells, we observed a decrease in Rho A activity as well as in the ratio of G/F actin on surfaces with either pits or posts but despite this low activity, knockdown of Rho A led to a significant decrease in confined motility suggesting that while Rho A activity is reduced on these surfaces it still plays an important role in controlling cellular response to barriers. In SW620 cells, we observed that Rho A activity was greatest in cells plated on a post microtopography which led to increased cell motility, and an increase in actin cytoskeletal turnover

Loss of Mll3 Catalytic Function Promotes Aberrant Myelopoiesis

<div><p>Two of the most common myeloid malignancies, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), are associated with exceedingly low survival rates despite recent therapeutic advances. While their etiology is not completely understood, evidence suggests that certain chromosomal abnormalities contribute to MDS and AML progression. Among the most frequent chromosomal abnormalities in these disorders are alterations of chromosome 7: either complete loss of one copy of chromosome 7 (-7) or partial deletion of 7q (del(7q)), both of which increase the risk of progression from MDS to AML and are associated with chemoresistance. Notably, 7q36.1, a critical minimally deleted region in 7q, includes the gene encoding the histone methyltransferase mixed-lineage leukemia 3 (<i>MLL3</i>), which is also mutated in a small percentage of AML patients. However, the mechanisms by which <i>MLL3</i> loss contributes to malignancy are unknown. Using an engineered mouse model expressing a catalytically inactive form of Mll3, we found a significant shift in hematopoiesis toward the granulocyte/macrophage lineage, correlating with myeloid infiltration and enlargement of secondary lymphoid organs. Therefore, we propose that <i>MLL3</i> loss in patients may contribute to the progression of MDS and AML by promoting myelopoiesis.</p></div

Impact of Mll3 loss-of-function on hematopoietic progenitor populations.

<p>Shown is <b>A</b>, the total number of BM cells, and the percentages of <b>B</b>, B cells and <b>C</b>, Gr1⁺ Mac1⁺ myeloid cells, where each point on the graph represents one mouse. Values in panels <i>B</i> and <i>C</i> are expressed as percentages of total cells. <b>D</b>, Representative H&E stains from one individual <i>Mll3</i>^+/+ and <i>Mll3</i>^Δ/Δ mouse of sternum sections taken at 400X magnification. Images depict the entire field of view (top panels) and a zoomed-in view (bottom panels). Scale bars are 50μm. <b>E-I</b>, Similar to panels <i>B</i> and <i>C</i>, except that the percentages of LSK cells, CLP, CMP, MEP, and GMP are shown, respectively. <b>B, C, I</b>, *p<0.05, **p<0.01, ***p<0.005 as determined by the Student’s <i>t</i>-test. <b>G</b>, *p<0.05 as determined by the Mann-Whitney test.</p

Phenotypic characterization of mutant <i>Mll3</i> mice.

<p><b>A</b>, Spleen weight expressed as a percentage of total mouse weight and <b>B</b>, the combined weight of four LN normalized to total mouse weight. Each point represents one mouse, and graphs include a combination of female and male organ weights. <b>C</b>, Similar to panel <i>A</i>, except liver weight is shown. <b>D</b>, Female mouse weight, where each point represents one mouse. <b>E</b>, Similar to panel <i>D</i>, except male mouse weight is shown. <b>F</b>, Similar to panels <i>A</i> and <i>C</i>, except kidney weight is shown. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001 as determined by the Mann-Whitney test.</p

Quantification and distribution of immune cells in the LNs of <i>Mll3</i> mice.

<p>Shown is the percentage of total cells (top) and absolute number (bottom) of <b>A</b>, B cells, <b>B</b>, T cells, and <b>C</b>, Gr1⁺ Mac1⁺ myeloid cells. <b>D</b>, Similar to panels <i>A-C</i>, except the total numbers of CD4⁺ (left) and CD8⁺ (right) T cells are shown. Each point represents one mouse. <b>B-C</b>, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 as determined by the Student’s <i>t</i>-test. <b>D</b>, *p<0.05, **p<0.005 as determined by the Mann-Whitney test. <b>E</b>, Representative H&E stains (top panels) and IHC for CD3 (bottom panels) from two individual <i>Mll3</i>^+/+ and <i>Mll3</i>^Δ/Δ mice of LN sections taken at 50X magnification. Scale bars are 500μm.</p

Relative <i>Mll3</i> expression in mouse hematopoietic cells.

<p>Absolute gene expression profiling adapted from Gene Expression Commons evaluating <i>Mll3</i> expression across 39 different hematopoietic cell populations in the mouse BM, spleen, and thymus. Shown are the normalized values of <i>Mll3</i> expression activity for several cell populations from 2–4 biological replicates. Positive values indicate high <i>Mll3</i> expression compared to the common reference, and negative values denote relatively low <i>Mll3</i> expression in comparison to the common reference. Error bars represent mean + SD. HSC, hematopoietic stem cell; Gra, granulocyte; Fr, fraction; Mz, marginal zone; Fo, follicular.</p

The extracellular matrix microtopography drives critical changes in cellular motility and Rho A activity in colon cancer cells

Abstract We have shown that the microtopography (mT) underlying colon cancer changes as a tumor de-differentiates. We distinguish the well-differentiated mT based on the increasing number of "pits" and poorly differentiated mT on the basis of increasing number of "posts." We investigated Rho A as a mechanosensing protein using mT features derived from those observed in the ECM of colon cancer. We evaluated Rho A activity in less-tumorogenic (Caco-2 E) and more tumorigenic (SW620) colon cancer cell-lines on microfabricated pits and posts at 2.5 μm diameter and 200 nm depth/height. In Caco-2 E cells, we observed a decrease in Rho A activity as well as in the ratio of G/F actin on surfaces with either pits or posts but despite this low activity, knockdown of Rho A led to a significant decrease in confined motility suggesting that while Rho A activity is reduced on these surfaces it still plays an important role in controlling cellular response to barriers. In SW620 cells, we observed that Rho A activity was greatest in cells plated on a post microtopography which led to increased cell motility, and an increase in actin cytoskeletal turnover.</p

UTX/KDM6A Loss Enhances the Malignant Phenotype of Multiple Myeloma and Sensitizes Cells to EZH2 inhibition

Loss or inactivation of the histone H3K27 demethylase UTX occurs in several malignancies, including multiple myeloma (MM). Using an isogenic cell system, we found that loss of UTX leads to deactivation of gene expression ultimately promoting the proliferation, clonogenicity, adhesion, and tumorigenicity of MM cells. Moreover, UTX mutant cells showed increased in vitro and in vivo sensitivity to inhibition of EZH2, a histone methyltransferase that generates H3K27me3. Such sensitivity was related to a decrease in the levels of IRF4 and c-MYC and an activation of repressors of IRF4 characteristic of germinal center B cells such as BCL6 and IRF1. Rebalance of H3K27me3 levels at specific genes through EZH2 inhibitors may be a therapeutic strategy in MM cases harboring UTX mutations