Unlocking Drug Resistance in Multiple Myeloma: Adipocytes as Modulators of Treatment Response.

Multiple myeloma (MM) is an incurable hematological malignancy characterized by the clonal proliferation of malignant plasma cells. Despite the development of a diverse array of targeted drug therapies over the last decade, patients often relapse and develop refractory disease due to multidrug resistance. Obesity is a growing public health threat and a risk factor for multiple myeloma, although the mechanisms by which obesity contributes to MM growth and progression have not been fully elucidated. In the present study, we evaluated whether crosstalk between adipocytes and MM cells promoted drug resistance and whether this was amplified by obesity. Human adipose-derived stem cells (ASCs) from nineteen normal (BMI = 20-25 kg/m2), overweight (25-30 kg/m2), or obese (30-35 kg/m2) patients undergoing elective liposuction were utilized. Cells were differentiated into adipocytes, co-cultured with RPMI 8226 or U266B1 multiple myeloma cell lines, and treated with standard MM therapies, including bortezomib or a triple combination of bortezomib, dexamethasone, and lenalidomide. We found that adipocytes from overweight and obese individuals increased cell adhesion-mediated drug resistance (CAM-DR) survival signals in MM cells, and P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) drug transporter expression. Further, co-culture enhanced in vitro angiogenesis, MMP-2 activity, and protected MM cells from drug-induced decreases in viability. In summary, we provide an underlying mechanism by which obesity can impair the drug response to MM and allow for recurrence and/or disease progression.

RUNX1 mutations mitigate quiescence to promote transformation of hematopoietic progenitors in Fanconi anemia.

Many inherited bone marrow failure syndromes (IBMFSs) present a high risk of transformation to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). During transformation of IBMFSs, hematopoietic stem and progenitor cells (HSPCs) with poor fitness gain ectopic, dysregulated self-renewal secondary to somatic mutations via undefined mechanisms. Here, in the context of the prototypical IBMFS Fanconi anemia (FA), we performed multiplexed gene editing of mutational hotspots in MDS-associated genes in human induced pluripotent stem cells (iPSCs) followed by hematopoietic differentiation. We observed aberrant self-renewal and impaired differentiation of HSPCs with enrichment of RUNX1 insertions and deletions (indels), generating a model of IBMFS-associated MDS. We observed that compared to the failure state, FA MDS cells show mutant RUNX1-mediated blunting of the G1/S cell cycle checkpoint that is normally activated in FA in response to DNA damage. RUNX1 indels also lead to activation of innate immune signaling, which stabilizes the homologous recombination (HR) effector BRCA1, and this pathway can be targeted to abrogate viability and restore sensitivity to genotoxins in FA MDS. Together, these studies develop a paradigm for modeling clonal evolution in IBMFSs, provide basic understanding of the pathogenesis of MDS, and uncover a therapeutic target in FA-associated MDS.

Endothelial Cell Tube Formation Assay: An In Vitro Model for Angiogenesis.

Endothelial cell tube formation assay is one of the most widely used and reliable methods for studying in vitro angiogenesis. Endothelial cells plated over a basement membrane extract and subjected to angiogenic factors in conditioned medium, form a rapid and quantifiable tube network within hours. Tube formation is sustained for 18-24 h, after which time apoptosis occurs and tube networks disintegrate. The tube network can be imaged using a phase contrast microscope, or upon Calcein-AM treatment, a fluorescence/confocal microscope. This assay has several advantages, namely: ease of set up, the ability to test numerous angiogenic/anti-angiogenic factors simultaneously, quick network formation, ability to view live or fixed tube networks, and quantifiability. To ensure successful results and limit variability, proper selection of basement membrane extracts and endothelial cells is necessary, and conditions must be optimized. In summary, this assay is a useful method for screening potential angiogenic/anti-angiogenic factors as well as identifying critical mechanisms and signaling pathways underlying angiogenic-related pathologies.

Loss of Tpl2 activates compensatory signaling and resistance to EGFR/MET dual inhibition in v-RAS transduced keratinocytes.

Cutaneous squamous cell carcinoma (cSCC) is the second most common form of skin cancer in the United States, affecting one million people per year. Patients with aggressive disease have limited treatment options and high mortality, highlighting the need to identify new biomarkers linked to poor clinical outcome. HRAS mutations are found in skin papillomas and cSCCs and increase in frequency when MAP3K family members are inhibited, suggesting a link between blockade of mitogen-activated protein kinase (MAPK) signaling and initiation of RAS-primed cells. Tpl2, a MAP3K gene, can serve as a tumor suppressor gene in cSCC. We have previously shown that upon Tpl2 ablation, mice have heightened sensitivity to aberrant RAS signaling. Tpl2-/- mice display significantly higher numbers of papillomas and cSCCs in two-stage chemical carcinogenesis studies and increased tumorigenicity of keratinocytes expressing oncogenic v-rasHa in nude mouse skin grafts. In part, this is mediated through increased mesenchymal-epithelial transition factor (MET) receptor activity. Epidermal Growth Factor Receptor (EGFR) is reported to be an essential factor for MET-driven carcinogenesis and MET activation may confer resistance to EGFR therapies, suggesting that the concurrent use of both an EGFR inhibitor and a MET inhibitor may show promise in advanced cSCCs. In this study we assessed whether normal or Ras-transformed Tpl2-/- keratinocytes have aberrant EGFR signaling and whether concomitant treatment with EGFR/MET tyrosine kinase inhibitors was more effective than single agents in reducing growth and angiogenic potential of Ras-transformed keratinocytes. Tpl2-/- keratinocytes exhibited increased HER-2 and STAT-3 under basal conditions and elevated p-MET and p-EGFR when transduced with oncogenic RAS. Inhibition of MET by Capmatinib increased p-EGFR in Tpl2-/- keratinocytes and papillomas, and inhibition of EGFR by Gefitinib increased HER2 and HER3 signaling in both genotypes. Treatment of keratinocytes with EGFR and MET inhibitors, in combination, significantly enhanced endothelial tube formation, MMP-9 activity and activation of other RTKs, with more pronounced effects when Tpl2 was ablated. These data indicate that Tpl2 cross-talks with both EGFR and MET signaling pathways. Upon inhibition of EGFR/MET signaling, a myriad of escape mechanisms exists in keratinocytes to overcome targeted drug effects.