Impact of pinworm infection on the development of murine B-cell leukemia/lymphoma in the presence and absence of ETV6::RUNX1.

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Resistance to mTOR Kinase Inhibitors in Lymphoma Cells Lacking 4EBP1

<div><p>Inhibitors of the mechanistic target of rapamycin (mTOR) hold promise for treatment of hematological malignancies. Analogs of the allosteric mTOR inhibitor rapamycin are approved for mantle cell lymphoma but have limited efficacy in other blood cancers. ATP-competitive “active-site” mTOR inhibitors produce more complete mTOR inhibition and are more effective than rapamycin in preclinical models of leukemia, lymphoma and multiple myeloma. In parallel to clinical trials of active-site mTOR inhibitors, it will be important to identify resistance mechanisms that might limit drug efficacy in certain patients. From a panel of diffuse large B-cell lymphoma cell lines, we found that the VAL cell line is particularly resistant to apoptosis in the presence of active-site mTOR inhibitors. Mechanistic investigation showed that VAL does not express eukaryotic initiation factor 4E-binding protein-1 (4EBP1), a key negative regulator of translation controlled by mTOR. Although VAL cells express the related protein 4EBP2, mTOR inhibitor treatment fails to displace eukaryotic initiation factor 4G from the mRNA cap-binding complex. Knockdown of eukaryotic initiation factor 4E, or re-expression of 4EBP1, sensitizes cells to apoptosis when treated with active-site mTOR inhibitors. These findings provide a naturally occurring example of 4EBP deficiency driving lymphoma cell resistance to active-site mTOR inhibitors.</p></div

Decreased IL-10 accelerates B-cell leukemia/lymphoma in a mouse model of pediatric lymphoid leukemia.

Exposures to a wide repertoire of common childhood infections and strong inflammatory responses to those infections are associated with the risk of pediatric B-cell acute lymphoblastic leukemia (B-ALL) in opposing directions. Neonatal inflammatory markers are also related to risk by unknown mechanism(s). Here, we demonstrate that interleukin-10 (IL-10) deficiency, which is associated with childhood B-ALL, indirectly impairs B lymphopoiesis and increases B-cell DNA damage in association with a module of 6 proinflammatory/myeloid-associated cytokines (IL-1α, IL-6, IL-12p40, IL-13, macrophage inflammatory protein-1β/CCL4, and granulocyte colony-stimulating factor). Importantly, antibiotics attenuated inflammation and B-cell defects in preleukemic Cdkn2a-/-Il10-/- mice. In an ETV6-RUNX1+ (E6R1+) Cdkn2a-/- mouse model of B-ALL, decreased levels of IL-10 accelerated B-cell neoplasms in a dose-dependent manner and altered the mutational profile of these neoplasms. Our results illuminate a mechanism through which a low level of IL-10 can create a risk for leukemic transformation and support developing evidence that microbial dysbiosis contributes to pediatric B-ALL

Expressing 4EBP1 in the VAL cells restores the effects of asTORi on cap dependent translation.

<p>m⁷-GTP pull down assay to measure the levels of cap bound proteins after a 4 hour MLN0128 treatment in the VAL and OCI-LY7 cells expressing 4EBP1 or the empty vector (EV) control. 4EBP1 expression was induced with 1 µg/ml Doxycycline for 16 hrs followed by a 4 hr treatment with 100 nM MLN0128. Results are representative of two independent experiments.</p

4EBP1 expression in the VAL cells causes asTORi mediated decrease in cap dependent translation and sensitizes them to MLN0128.

<p>a. Luciferase reporter assays in VAL and OCI-LY7 cells expressing 4EBP1. Cells were electroporated with the dual luciferase reporter constructs and treated with 100 nM MLN0128 (+/−1 µg/ml Doxycycline) for 16 hrs. Results are averaged for three independent experiments and statistical significance measured using a t-test (*p<0.05, **p<0.01). b. Propidium iodide staining to measure the percentage of cells with sub-diploid DNA content in the VAL cells expressing 4EBP1 upon treatment with 100 nM MLN0128 with and without doxycycline induction. Results averaged for three independent experiments. Statistical analysis using a t-test (**p<0.01). c. Annexin V and PI staining to measure the percentage of cells undergoing apoptosis in the VAL cells expressing 4EBP1 upon treatment with 100 nM MLN0128 with and without doxycycline induction for 48 hours. Results averaged for three independent experiments. Statistical analysis using a t-test (**p<0.01). Shown to the right of the graphs is a western blot showing induction of 4EBP1 expression with 1 µg/ml Doxycycline.</p

VAL cells lack 4EBP1.

<p>Western blot analysis of total cell lysates from DLBCL cell lines after a 4</p

VAL cells are resistant to disruption of the translationally active eIF4F complex by asTORi.

<p>m⁷-GTP pull down assays of VAL and OCI-LY1 cells treated with 100 nM MLN0128, 500 nM PP242 or 10 nM rapamycin for 4 hours. Proteins from the cap bound fraction were detected by western blotting along with the total cellular proteins. Results are representative of several independent experiments.</p

Differential sensitivity of DLBCL cell lines to asTORi.

<p>a. Annexin V and propidium iodide staining to assess DLBCL cell lines undergoing apoptosis following a 48* p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001. b. Cell cycle analysis after a 48 hour treatment with the indicated inhibitors. Results graphed as a percentage of cells in G1 phase. n = 3, error bars represent SEM. * p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001.</p