PS1363 TARGETING MITOCHONDRIAL FITNESS OF MYELOMA CELLS WITH TAK‐242 OVERCOMES BORTEZOMIB RESISTANCE
Authors: Giallongo C. , Tibullo D. , Giuseppina C. , Puglisi F. , Cambria D. , Parrinello N. , Romano A. , Conticello C. , Volti G. Li , Palumbo G.A. , Di Raimondo F. 
Published: June 2019

Background:

Multiple myeloma (MM) is a B‐cell malignancy critically dependent for survival and proliferation on signals coming from its inflammatory microenvironment and toll‐like receptors (TLR) may greatly contribute to inflammation. TLR are expressed on human MM cells and in particular TLR4 ligand promotes proliferation of these malignant cells and immune escape mechanisms.

Aims:

Since it has been demonstrated that the acquisition of proteasome inhibitors (PI) resistance involved increased mitochondrial respiration, we investigated the possible implication of TLR4 signaling with mitocondrial fitness as potential mechanism of drug resistance.

Methods:

Flow cytometry was used to evaluate apoptosis, ROS levels, mitochondrial depolarization (DiOC2(3) staining) and mitochondrial mass (Mitotracker staining).

Results:

The activation of TLR4 signaling by LPS treatment induced mitochondrial biogenesis by increasing TFAM expression and mitochondrial mass in MM cells; this effect was reverted by the treatment with a TAK‐242, a selective inhibitor of TLR4 signaling.

Bortezomib (BTZ) treatment induced over‐expression of TLR4 (p < 0.001) in MM cell lines (U266, MM1S, OPM2, H929) and its signaling was functional as suggested by up‐regulation of MyD88 and MAPK activation. TLR4 protein was over‐expressed in Bortezomib‐resistant U266 (U266‐R) cells compared to bortezomib‐sensitive U266 (U266‐S) cells. Proteasome activity assay revealed that BTZ was still able to inhibit proteasome in U266‐R in the same way as U266‐S. U266‐R showed higher mitochondrial mass and up‐regulated TFAM (p < 0.01).

To investigate whether TLR4 was involved in resistance to BTZ going through mitochondrial fitness, U266‐R cells were treated with either 15 nM BTZ, 20 μM TAK‐242 or their combination. Combinatorial treatment overcame resistance inducing 55% of apoptosis.

Taking into account that oxidative stress is an important mechanism of PI cytotoxicity, we evaluated ROS. Compared to BTZ alone, TAK‐242/BTZ treated cells showed higher and extended ROS levels, have less mitochondrial mass and more depolarized mitochondria (p < 0.001). Moreover, BTZ alone induced over‐expression of NDUFA6 and ND4 (subunits of mitochondrial respiratory complex I), TFAM, OPA1 and MNF2 (important in fusion process); on the contrary their expression decreased after combination with TAK‐242 (p < 0.001).

Increased ROS and mitochondrial depolarization activate mitophagy. Therefore, we evaluated co‐localization of mitochondria (stained with MitoTracker) with the autophagosome marker LC3‐II using confocal microscopy. BTZ and TAK‐242/BTZ increased Mitotracker/LC3‐II co‐localization respectively of about 4,5 and 50 fold compared with control.

It is known that cells can activate SQSTM1 to promote mitochondrial aggregation and avoid apoptosis triggered by mitochondrial depolarization. Interestingly, TAK‐242/BTZ down‐regulated SQSTM1 expression sensitizing resistant cells to apoptosis through the disruption of mitochondrial aggregation and autophagic clearance of mitochondria.

Summary/Conclusion:

Our data demonstrated that TLR4 inhibition in bortezomib‐resistant cells impaired mitochondrial fitness resulting in apoptosis and overcoming of drug resistance.