The introduction of novel agents has improved the outcome of MM patients, but MM is still considered an incurable disease and the emergence of resistance is the main responsible for this situation. The unraveling of the resistance mechanisms would help to design novel therapeutic strategies (combinations or sequencing treatments) to overcome this problem.
Material and Methods
We have developed an in vivo model of acquired resistance to antimyeloma agents based in a model of subcutaneous plasmocytoma (MM1S) in CB17-SCID mice. For this purpose, mice were treated with Lenalidomide (25 mg/Kg) + Dex (1 mg/Kg) (LD), Pomalidomide (5 mg/Kg) + Dex (PD) or vehicle control, and after a period of initial sensitivity, tumors developed resistance to the administered combination. At this moment selected sensitive and resistant tumors were excised to analyze apoptosis, signaling pathways and gene expression profiling (GEP) changes. Moreover, some of these mice bearing resistant tumors were switched to receive the alternative combination (that is LD-PD or PD- LD), and, in selected mice, a second change of treatment was performed after secondary resistance to receive again the initial treatment (LD-PD-LD or PD-LD-PD). In order to evaluate TTP and to define the moment to change treatment we considered progression when tumor volume reached 1.700 mm3.
Both combinations (LD & PD) controlled the initial growth of the tumors, with a higher potency for the PD combination. Tumor volume reached 500 mm3 at a median of 8, 42 & 53 days for control, LD and PD respectively (p=0.01 and p=0.001). Nevertheless, after 30 days of continuous treatment, and despite maintaining the administration of the drugs, tumors started growing, and, once the tumors had reached 500 mm3, their growth kinetic was similar for the treated mice (despite still being treated) as compared to the untreated mice, indicating the emergence of complete resistance. This resistance was also confirmed ex vivo by in vitro culture with the corresponding drugs.
In order to test the presence of cross-resistance, mice bearing big tumors resistant to LD or PD and already growing in an exponential phase (volume of 1.700 mm3), were at this point treated with the alternative combination. This sequential treatment change induced tumor stabilization and even a decrease of tumor volume. Again PD was significantly more potent at overcoming LD resistance as compared to the alternative situation, and this was verified both in terms of tumor growth inhibition (p=0.005) and in terms of time to progression (median of 16 vs 27 days for LD and PD respectively. p=0.004).
Furthermore, mice that had been treated with LD-PD or PD-LD and had developed resistance to both combinations were again treated with the initial combination, and, surprisingly, they were again sensitive, indicating the reversibility of the acquired resistance. Similarly to previous experiments, PD was again significantly more potent than LD. This reversibility was also confirmed ex vivo after culture of the cells in medium without drugs.
In order to investigate the resistance mechanisms, cells extracted from sensitive and resistant tumors were analyzed by Western Blot. Treatment with LD and PD induced a downregulation of pERK 1/2, nevertheless, when these cells developed resistance a very significant increase of pERK 1/2 levels (even higher than the basal levels) was observed. Moreover, these resistant cells also showed an upregulation of p-MEK, p-RAF and RAS. In this same line, the MEK inhibitor PD-98059 potentiated the in vitro activity of LD or PD in fresh MM1S cells, with high synergism (CI<0.1).
Finally, changes in GEP were evaluated in extracted tumors. A significant change was observed in cells from tumors sensitive to PD and LD (with a higher gene deregulation with the former combination), as compared to untreated ones. Interestingly, when tumors became resistant, most of these changes disappeared and the GEP partially returned to a profile similar to that of active untreated tumors.
The data presented would support treatment with alternative IMIDs if resistance was developed to one of them (specially PD was significantly more potent at rescuing resistance to LD), or even the retreatment with the same IMID after a wash up period. Moreover this study supports the evaluation of combinations of IMIDs with agents that abrogate the ERK pathway in order to increase efficacy or avoid resistance.