Page 163 - Haematologica Vol. 110 - January 2025

ARTICLE - Targeted CDK7 and BRD4 inhibition in myeloma
Y. Yao et al.
superior activity of the combination against all MM cell lines tested compared to single perturbation alone (Figure 1A and Online Supplementary Figure S1A). Isobologram analysis revealed strong synergism of the combination as compared to single agents, with a combination index <1.0 at all tested doses (Figure 1B). On the other hand, peripheral blood mononuclear cells from healthy donors were less sensitive to the combination therapy (Online Supplementary Figure S1B). We also tested the effect of the combination in XG1 MM cells, which have intrinsic resistance to both YKL-5-124 and JQ1 and confirmed dual inhibition to be synthetically lethal in these cells (Figure 1C, D and Online Supplementary Figure S1C). These find- ings were validated with two additional BET inhibitors: ARV-825 and I-BET151 (Figure 1E, F).
Consistent with YKL-5-124 being a CDK7 inhibitor, we ob- served reduced phosphorylation of CDK4 and increased activation of RB with both YKL-5-124 single agent and combination therapy (Figure 2A, B), this latter causing early cell cycle arrest, with a higher percentage of cells in the G1 phase than with either agent alone (Figure 2C) and a moderate increase in apoptotic cell death and cleavage of caspase 3 (Figure 2D and Online Supplementary Figure S1D). In addition, we tested the combination of JQ1 with the Food and Drug Administration-approved CDK4/6 inhibitor palbociclib, confirming its greater anti-MM effect in this context than when administered as a single drug (Online Supplementary Figure S1E). The combination of low doses of JQ1 and palbociclib was indeed synergistic, with isobolo- graphic analysis revealing a strong synergism in most MM cell lines (Online Supplementary Figure S1F). This synergism between BRD4 and cell cycle inhibitors was also observed in other cancer contexts.22-24
We next assessed whether this combination regimen could be applicable to other hematologic malignancies, such as WM. We observed that WM cells are more sensitive to the inhibition of CDK7 than to BET (Online Supplementary Figure S2A). Indeed, CDK7 inhibition robustly reduced the viability of WM cell lines (N=3) and primary WM cells in a dose- and time-dependent manner (Online Supplementary Figure S2B), impairing E2F, MYC and cell cycle–associated gene set signatures, as observed in MM cells (Online Sup- plementary Figure S2C). Moreover, combination treatment effectively killed WM cell lines and primary cells from pa- tients, although the effect was greater in MM cells (Online Supplementary Figure S2D, E).
Combination therapy alters the tumor cell transcriptome
To gain deeper insight into the molecular basis of the observed drug synergism, we performed gene expression profiling after 24 hours of treatment with 200 nM JQ1 and 50 nM YKL-5-124 in the AMO1 cell line. RNA-sequencing analysis of cells treated with a low dose of YKL-5-124, a low dose of JQ1, or a combination showed that, in gener- al, combination therapy had a more profound impact on
the transcriptome than single-agent therapy (Figure 3A). Canonical pathway analysis revealed that the upregulated pathways were enriched in pathways for autophagy and senescence, whereas cell cycle regulation, glycolysis, the unfolded protein response, and cholesterol biosynthesis were enriched among the downregulated pathways (Figure 3B). Moreover, analysis of the predicted transcriptional reg- ulators found that FOXM1, BRD4, E2F and MYC were among the inhibited regulators while CDKN1A(p21), TP53 and RB were among the activated regulators (Figure 3C).
In line with this result, we observed a pronounced decrease in the expression of MYC upon combination therapy (Figure 3D). Moreover, dual targeting synergistically decreased the expression of additional genes important to the MM onco- genic state, including MYBL2 (b-Myb) (Figure 3E). MYBL2 is a cell-cycle and proliferation gene frequently overexpressed in several cancers: here, we found MYBL2 to be a depen- dency in a panel of MM cell lines (Online Supplementary Figure S3A), with its high expression associated with poor outcome in datasets of MM patients (Online Supplementary Figure S3B, C).
CDK7 inhibition synergizes with JQ1 against multiple myeloma cells ex vivo and in vivo
We finally tested the therapeutic potential of the various drug combinations. We first confirmed the strong syner- gism observed in primary MM cells, where a combination index <1.0 was found at all tested doses, with the effect being considerably less significant in peripheral blood mononuclear cells derived from MM patients (Figure 4A). This was also confirmed in primary cells treated with a combination of JQ1 and palbociclib (Online Supplemen- tary Figure S4A, B). We next evaluated the effect of the combination in the context of the bone marrow milieu. The combination of low doses of YKL-5-124 and JQ1 had a striking effect in inhibiting the proliferation of MM cells cultured for 24 hours with supernatant from bone marrow stromal cells derived from MM patients (Figure 4B). Sim- ilarly, the combination regimen had a significant impact on the viability of primary CD138+ MM cells cultured in the presence of their bone marrow microenvironment, while CD138– bone marrow cells derived from the same patient were unaffected (Figure 4C). Altogether these data suggest a favorable therapeutic index.
The synergistic effects of combining YKL-5-124 with JQ1 in vitro prompted us to assess the efficacy of this com- bination in two in vivo models of MM. SCID mice were injected subcutaneously with MM1s or H929 cells. When tumors reached 100 mm3, the mice were randomized to vehicle or a low dose of JQ1 (50 mg/kg) or YKL-5-124 (2.5 mg/kg) or combination treatment for 2 weeks. Combination therapy resulted in smaller tumors compared to those in the animals treated with the single agents (Figure 4D and Online Supplementary Figure S4C), without evident toxicity or weight loss (Figure 4E). This pattern was also
Haematologica | 110 January 2025
155