**3. Immunomodulatory agents**

The immunomodulatory drugs (IMiDs), Thalidomide, Lenalidomide and Pomalidomide have also made a major impact in the management of MM. Despite a checkered history in the 1950s and 1960s due to teratogenicity, Thalidomide has high anti-MM activity and has been incorporated into many treatment regimens. The second generation IMiD Lenalidomide and third generation IMiD Pomalidomide represent sequential improvements in efficacy and toxicity profiles with demonstrable activity in patients who have developed resistance to an earlier generation IMiD [96]. With regard to Lenalidomide, the MM-009 [97] and MM-010 [98] phase III trials demonstrated the superiority of Lenalidomide and Dexamethasone over Dexamethasone in relapsed/refractory MM patients whilst the pivotal MM-003 study [99] demonstrated the efficacy of Pomalidomide and Dexamethasone in MM patients who were refractory to both Bortezomib and Lenalidomide.

The anti-MM effects of IMiDs are related to their binding to the E3 ubiquitin ligase cereblon (CRBN) and subsequent ubiquitination and degradation of two B-cell transcription factors, Ikaros (IKZF1) and Aiolos (IKZF3) [96]. A landmark study identified CRBN as a primary target in Thalidomide teratogenicity, further demonstrating that Thalidomide binds to CRBN, disrupting the function of the E3 ubiquitin ligase complex, ultimately leading to the downregulation of fibroblast growth factor genes and the teratogenic effects associated with Thalidomide [100]. Subsequently, it was shown that the anti-MM efficacy of IMiDs is directly related to CRBN expression.

#### **3.1. Mechanisms of resistance to immunomodulatory agents**

#### *3.1.1. Pre-clinical/clinical findings*

Bortezomib resistance in MM patients [91] whilst suppression of miR-15a and -16 by BMSCs was shown to be responsible for the protection of MM cells from Bortezomib-induced apoptosis [91]. miR-29 acts as a tumour suppressor miRNA and is downregulated in patient MM cells and in MM cell lines with acquired resistance to Bortezomib, Carfilzomib and Ixazomib [91]. Finally, exosomes mediate local cell-cell signalling by transferring mRNAs, miRNAs and proteins. It has been shown that exosomes derived from BMSCs inhibited Bortezomib-

In a phase II study, the anti-IL-6 antibody Siltuximab was administered with Dexamethasone to patients with relapsed and/or refractory MM [93]. Although no responses to Siltuximab alone were observed, the addition of Dexamethasone resulted in ORR, PFS and OS of 23%, 3.7 months and 20.4 months, respectively. Despite these findings, this strategy has not progressed further. The c-MET inhibitor Tivantinib was examined as a single agent in a phase II study in relapsed/refractory MM patients [94]. Overall, 36% of patients showed stable disease as their best response with the authors concluding that Tivantinib did not show promise for unselected relapsed/refractory MM patients, however, the fact that a significant proportion did show disease stability suggests combining c-MET inhibition with other anti-MM therapy could be explored. There are a small number of phase I studies employing a monoclonal anti-IGF-1R antibody alone or in combination with Bortezomib in relapsed/refractory MM, however, the authors of one study conclude that due to low response rates, even in combination with Bortezomib, further development is not justified [95]. Note should be made that patient recruitment into this study was not performed based on evaluation of IGF-1R expression on patient MM cells. No small molecule inhibitors of IGF-1R have so far been tested clinically. A phase I clinical trial in relapsed/refractory MM patients suggests that AKT inhibition with Afuresertib might overcome resistance to Bortezomib [87]. In this study, the ORR was 8.8%, however, despite these potentially promising results in heavily pre-treated patients, more advanced clin-

The immunomodulatory drugs (IMiDs), Thalidomide, Lenalidomide and Pomalidomide have also made a major impact in the management of MM. Despite a checkered history in the 1950s and 1960s due to teratogenicity, Thalidomide has high anti-MM activity and has been incorporated into many treatment regimens. The second generation IMiD Lenalidomide and third generation IMiD Pomalidomide represent sequential improvements in efficacy and toxicity profiles with demonstrable activity in patients who have developed resistance to an earlier generation IMiD [96]. With regard to Lenalidomide, the MM-009 [97] and MM-010 [98] phase III trials demonstrated the superiority of Lenalidomide and Dexamethasone over Dexamethasone in relapsed/refractory MM patients whilst the pivotal MM-003 study [99] demonstrated the efficacy of Pomalidomide and Dexamethasone in MM patients who were

induced cell death to protect MM cells from apoptosis [92].

*2.3.7.2. Clinical studies to circumvent resistance*

78 Update on Multiple Myeloma

ical trials have not been undertaken.

**3. Immunomodulatory agents**

refractory to both Bortezomib and Lenalidomide.

Resistance mechanisms to IMiDs have been elucidated to a far lesser extent than have those for proteasome inhibitors (**Table 1** and **Figure 1B**) and mostly hinge on the presence of functional CRBN in MM cells [100]. MM patients exposed to and thought to be resistant to Lenalidomide had lower CRBN levels compared to paired samples before and after therapy [101]. Subsequently, it was shown that high expression of CRBN is associated with a favourable response to Thalidomide and Lenalidomide in newly-diagnosed MM patients [102, 103] and no IMiD response occurred in patients with very low CRBN levels [104]. Moreover, in MM patients refractory to Pomalidomide, CRBN levels predicted for differences in PFS (3 versus 8.9 months) and OS (9.1 versus 27.2 months) when comparing patients in the lowest CRBN expression quartile versus those with higher expression [104]. Notably, as CRBN expression decreases in MM patients who develop resistance to Lenalidomide therapy, this does not affect sensitivity to Bortezomib, Melphalan and Dexamethasone [101, 105]. Low levels of the CRBN binding protein IKZF1 and high levels of another CRBN binding protein Karyopherin Subunit Alpha 2 (KPNA2) also correlated with lack of response to Pomalidomide and/or OS [106]. Specifically, patients with low IKZF1 expression had a median OS of 7.3 months compared with 27.2 months in those with higher IKZF1 expression which was also correlated with a similar pattern of PFS (4.9 vs. 7.3 months) [106].

In relapsed/refractory MM patients, the majority (88%) of whom were refractory to an IMiD, an increased prevalence of mutations in the Ras pathway genes KRAS, NRAS and/ or BRAF (72%), as well as TP53 (26%), CRBN (12%) and CRBN pathway genes (10%) were observed [107]. Notably, all CRBN-mutated patients and 91% of the CRBN pathway-mutated patients were unresponsive to IMiD based treatment. Moreover, three patients with CRBN mutations at the time of IMiD resistance did not possess these genetic aberrations at the time of IMiD sensitivity. Importantly, the introduction of these mutations in MM cells conferred Lenalidomide resistance *in vitro* [107]. Finally, a pre-clinical study has demonstrated that Lenalidomide resistant MM models over-express the hyaluronan (HA)-binding protein CD44, a downstream Wnt/β-catenin transcriptional target [108]. Consistent with this hypothesis, Lenalidomide resistant MM cell lines show greater adhesion to bone marrow stromal cells. Inhibition of CD44 by application of the humanised monoclonal anti-CD44 antibody RO5429083 induced a modest anti-proliferative effect whilst shRNA-mediated CD44 knockdown resulted in a marked re-sensitisation to Lenalidomide [108].

#### *3.1.2. Clinical studies to circumvent resistance*

Whilst the CRBN pathway has been shown to be pivotal in IMiD responsiveness, no clinical studies have eventuated that make use of this important biology as a strategy to overcome resistance to IMiDs and many questions remain such as how much functional CRBN is actually required to maintain IMiD sensitivity. Despite the controversies surrounding CRBN, activating mutations in Ras pathway components, such as KRAS G12D and BRAF V600E, could potentially be targeted with existing compounds in MM patients harbouring these mutations [109]. Such studies have not yet been conducted, although two patients with BRAF V600E positive relapsed/refractory MM achieved significant reductions in tumour burden when treated with the BRAF inhibitor Vemurafenib whilst a patient with highly resistant and rapidly progressive MM also harbouring the BRAF V600E mutation achieved a rapid and sustained response with dual BRAF and MEK inhibition [110].

**4.1. Mechanisms of resistance to monoclonal antibodies**

The relatively recent addition of mAbs to MM pharmacotherapy means there is a paucity of studies examining resistance mechanisms although these are now being explored with their increasing clinical use (**Table 1** and **Figure 1C**). Examination of CD38 expression on MM cells in 102 patients treated with Daratumumab monotherapy has been insightful [117]. With regard to the effect of Daratumumab on residual bone marrow plasma cells, two important points were clear from this analysis. Firstly, CD38 cell surface expression on plasma cells is highest before Daratumumab treatment and is significantly decreased during treatment. At the time of progressive disease, plasma cells isolated from the bone marrow of these patients exhibited low expression of CD38 suggesting Daratumumab therapy would be less effective, a finding corroborated previously [76]. Secondly, pre-treatment CD38 expression on the surface of MM cells was higher in patients who achieved at least a PR compared to those who did not. Recently, it was shown that Daratumumab-CD38 complexes and accompanying cell membrane are actively transferred from MM cells to monocytes and granulocytes in a process called trogocytosis that was also associated with reduced MM cell surface expression of CD49d, CD56 and CD138 [118]. However, Daratumumab-induced reductions in CD38 expression on MM cells occur in patients with deep and durable responses suggesting reductions in CD38 alone are not responsible for Daratumumab resistance [118]. Cell surface expression of the complement-inhibitory proteins, CD46, CD55 and CD59, was not associated with clinical response but significantly increased only at the time of disease progression. Furthermore, all*trans* retinoic acid increased CD38 expression whilst decreasing expression of CD55 and CD59 on MM cells from patients who developed Daratumumab resistance to approximately pre-

Resistance Mechanisms to Novel Therapies in Myeloma http://dx.doi.org/10.5772/intechopen.77004 81

treatment levels, resulting in enhancement of Daratumumab-mediated CDC [117].

morphisms [123] and even KIR and HLA genotypes [124].

patients with relapsed/refractory MM (NCT02751255).

*4.1.2. Clinical studies to circumvent resistance*

In addition to the cell surface expression of target antigens on MM cells, several other potential mechanisms of resistance to mAbs may be at play. Soluble forms of CD38 [119] and SLAM7 [120] may affect the efficacy of Daratumumab and Elotuzumab, respectively. Another potential mechanism of resistance is the development of neutralising antibodies to the therapeutic antibody. This phenomenon was noted in 39% of patients treated with single agent Elotuzumab resulting in more pronounced effects on serum Elotuzumab concentrations [121]. Furthermore, in the ELOQUENT-2 trial, 15% of patients developed anti-Elotuzumab antibodies on at least one occasion [115], however, antibodies directed against Daratumumab have to this day not been detected. Other factors that may contribute to the clinical efficacy of mAb therapy include the frequency and activity of effector immune cells [122], Fcγ receptor poly-

Whilst the mechanisms of resistance to mAbs are being elucidated, clinical studies specifically designed to overcome these biological processes are largely lacking with the exception of an ongoing phase I/II trial of Daratumumab in combination with all-*trans* retinoic acid for

*4.1.1. Pre-clinical/clinical findings*
