The Role of the ABL1/YAP1/P73 Axis in Prevention of DNA Damage-Mediated Apoptosis in Multiple Myeloma

Genome integrity plays a crucial role in the development of normal plasma cells to eliminate aberrant ones. Multiple myeloma (MM) is a plasma cell malignancy characterized by complex heterogeneous cytogenetic abnormalities. MM cells show constitutive DNA Damage Response (DDR) and activate compensatory mechanisms to prevent DNA-damage mediated apoptosis. Here we define the molecular mechanisms of these protective effects.

A panel of 15 MM cell lines was used. Blood and BM samples from healthy volunteers and MM patients were obtained after informed consent and subjected to Ficoll-Paque density sedimentation to get mononuclear cells (MNCs). Patient MM cells were isolated from BM MNCs by CD138-positive selection. Lentiviral delivery system was used for expression and knock-down of YAP1 in KMS-18, KMS-20, MM.1S and UTMC-2 MM cell lines. The biologic impact of YAP1 phenotype was evaluated using cell growth, viability and apoptosis assays.

We confirmed that a wide range of MM patient cells and MM cell lines have markers of constitutive DDR, including phosphorylation of H2A.X, ATM, ATR, Chk2 and Chk1, assessed by western blot analysis and immunofluorescence. However, these MM cells do not show basal level of apoptosis. Specifically, cleaved forms of caspase 3 and PARP are lacking in non-treated cells, and the absence of co-expression of cleaved caspase 3 with phospho-H2A.X by immunofluorescence confirms that phospho-H2A.X positive cells are viable cells. Since DDR is present in both p53-wild-type (wt) and p53-mutated cell lines, we examined whether ABL1/YAP1/p73 axis represents an alternative and crucial pathway to avoid DNA-damage mediated apoptosis. Indeed, ABL1 is up-regulated and predominantly localizes in the nucleus, a potential apoptotic stimulus, in MM cells, as assessed by western blot and immunofluorescence. To define if ABL1 nuclear localization was triggered by DDR, we treated MM cells with a specific ATM inhibitor (Ku55933) and a DNA damaging agent, doxorubicin. Inhibition of DDR in both p53 wt cell lines (MM.1S and H929) and p53 mutated cell lines (UTMC-2, JJN-3 and KMS-20) causes ABL1 cytoplasmic retention, while doxorubicin increases ABL1 nuclear translocation. Co-treatment with doxorubicin and ABL1 inhibitor STI-571 rescues MM cells from doxorubicin-mediated cell death. In particular, apoptotic cells decrease from 47.2% to 21.5% in U266, from 55.3% to 12.4% in MM.1S, and from 57.9% to 19.1% in UTMC-2 cells in response to combination treatment. To delineate the molecular mechanisms whereby MM cells repress ABL1 pro-apoptotic function, we focused on YAP1, a downstream target of the Hippo pathway involved in ABL1 cascade. We explored a large dataset of aCGH data on MM patients and discovered that YAP1 genomic locus (chr. 11q22) is deleted, associated with BIRC2 and BIRC3 in 11% of patients. This genetic abnormality was also found in KMS-18 and KMS-20 cell lines. Although YAP1 expression was normal in peripheral blood MNCs (PBMCs), its expression was decreased in the majority of patient MM cells and MM cell lines regardless of the presence of focal deletion. Importantly, low expression of YAP1 is associated with poor prognosis of MM patients. To further delineate the biologic significance of YAP1 in MM cells, we re-expressed pLENTI4-YAP1-EGFP in MM cell lines with either YAP1 deletion (KMS-18, KMS-20) or YAP1 low expression (MM.1S). We also silenced YAP1 using two different specific shRNAs in UTMC-2 MM cell line. As expected, YAP1 re-expression reduces cellular growth and increases apoptosis in all cell lines tested (25.7%, 37.1% and 32.3% apoptotic KMS-20, KMS-18 and MM.1S cells, respectively), condition that was further enhanced by doxorubicin treatment. Previous studies have shown that P73 is expressed in MM even though at low levels and we here show that they inversely correlate with YAP1 protein expression. Importantly, YAP1 re-expression increases p73 stability and promotes transcription of p73-target genes including BAX, PUMA and p21. In contrast, UTMC-2-YAP1^−/− cells show improved survival with lower levels of basal apoptosis and higher resistance to treatment with bortezomib or doxorubicin.

YAP1 mediates a strong apoptotic signal for MM cells. Thus, activation and/or overexpression of YAP1 represent a novel therapeutic strategy to improve outcome of patients with MM.

Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder and Scientific Founder, Scientific Founder and Scientific Founder Other.