Drug Resistant Myeloma Cells Cause More Severe Bone Destruction in Myeloma
Authors: Hao Mu , Gu Zhimin , Machin Reinaldo Franqui , Xu Hongwei , Thomas Gregory S. , Qiu Lugui , Tricot Guido J , Zhan Fenghuang 
Published: October 2019

Abstract
Background: Multiple myeloma (MM) is the second most common hematologic malignancy in the US and is characterized by osteolytic disease. More than 80% of MM patients have osteolysis, leading to in fractures, severe bone pain, spinal cord compression, and hypercalcemia, greatly compromising patient quality of life and resulting in mortality. While bone exists in a dynamic state between resorption and deposition, the molecular mechanisms underlying bone destruction in MM remain elusive. One contributing factor is heparanase (HPSE). In our work, we have previously shown that the expression of NEK2 and HPSE are closely correlated. We have also shown that high expression of chromosomal instability-related genes (e.g. NEK2) induces drug resistance and poor prognosis in MM. Though tumor burden is dramatically decreased in remission, bone destruction is poorly repaired, suggesting drug resistant MM cells may play a critical role in inducing bone destruction or in preventing the repair of bone disease in remission.
Materials and Methods: Gene expression profiling (GEP) data and bone lytic lesions detected by MRI were collected from 333 newly diagnosed MM patients from the Total Therapy 2 (TT2) clinical trial. MM cell lines with stable overexpression or knockdown of NEK2 and/or HPSE were created using lentiviral delivery. Protein and mRNA expression was assessed by immunoblotting and real-time PCR, respectively. TRAP staining was used to assess osteoclast differentiation. In vivo studies were performed in the KaLwRij 5TGM1 MM mouse model in 3 different groups: empty vector (EV), NEK2 overexpression (NEK2 OE), or NEK2 OE with HPSE shRNA. Bone density and imaging assessment was performed via X-ray and microCT scanning. HPSE, NFκB, and USP7 contributions were examined using the specific small molecule inhibitors SST0001, BSM-345541, and P5091, respectively.
Results: In correlating NEK2 expression with osteolytic lesion from 333 newly diagnosed MMs, we found the expression of NEK2 was significantly higher in MM patients with bone lytic lesion than those without lesions. NEK2 expression was also higher in patients with severe bone disease (7 or more lesions) than those fewer bone lytic lesions (< 7).
To determine if NEK2 causes bone lytic lesions in MM, we performed in vivo studies injecting NEK2 OE 5TGM1 cells into KaLwRij mice. MicroCT showed clearly that bone volume and thickness of trabecular bone were decreased significantly in mice injected with NEK2 OE cells compared to EV cells. Strikingly, treatment with the HPSE inhibitor SST0001 dramatically rescued bone mass loss induced by NEK2 overexpression. Histological TRAP staining of decalcified bone sections revealed a significant increase in the number of osteoclasts in the bone surface area in mice injected with NEK2 OE cells compared to EV-treated mice, while addition of the SST0001 significantly reduced the number of osteoclast.
Because osteoclast causes bone resorption, we examined the effect of applying conditioned media from MM cells with or without NEK2 overexpression to pre-osteoclast cells derived from MM patients, C57BL6 mice, and the pre-osteoclast RAW264.7 cell line. We found significantly increased mature osteoclasts (>3 nuclei) after culture with media collected from NEK2 MM cells compared to conditioned media collected from EV cells. Consistently, osteoclast differentiation-associated genes (i.e. CTSK, NFATc1, RANK, and TRAP) were upregulated in pre-osteoclasts cultured with NEK2 OE conditioned media. SST0001 suppressed osteoclast differentiation induced by NEK2, strongly suggesting that HPSE mediates NEK2 induced osteoclastogensis and bone destruction in MM. Mechanistic studies revealed that NEK2 directly binds to and activates USP7 resulting in deubiquitination of NFκB. Inhibition of NFκB decreased NEK2-induced HPSE expression induced by NEK2 at both the mRNA and protein level.
Conclusions: Our findings suggest that high NEK2 expression may be a biomarker for bone disease in MM. We demonstrate that a subpopulation of drug-resistant, NEK2-overexpressing MM cells leads to increased production and activity of HPSE, and increased osteoclast differentiation contributing to severe bone disease. Mechanistically, we show that NEK2 interacts with USP7, stabilizing NFκB and upregulating HPSE. Our results demonstrate drug resistant MM cells may not only promote tumor cell survival but also contribute to bone destruction in MM.

Disclosures
No relevant conflicts of interest to declare.