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T. Lewis et al.
In vivo systemic xenograft model of myeloma in NOD/SCID mice
NOD/SCID mice were sublethally irradiated prior to tail vein inoculation with the human myeloma cell line RPMI8226 (1x107 cells) to initiate tumor development. The date of inoculation was denoted as day 0. Intravenous treatment with vehicle only; 0.05% dimethylsulfoxide in saline (n=7) or 1 mg/kg of DC-1-192 (n=7) was started at day 5. Survival was evaluated from the first day of treatment until death.
Statistical analysis
All statistical analyses were performed using Graphpad Prism 6.0 software (Graphpad Software). The normal distribution of the data was established using the omnibus K2 test. Univariate comparisons were made using the Student t-test for paired and unpaired observations. All toxicity data from drug treatment were used to produce sigmoidal dose-response curves from which LD50 values were calculated. Toxicity data from synergy experiments were processed using CalcuSyn software with the median effect method to subsequently calculate the combina- tion index (CI) for each pair of agents; CI values less <1 were indicative of synergy.22
Results
Cytotoxic screening of pyrrolo[2,1-c][1,4]benzodi- azepine compounds identified three lead compounds
Initial cytotoxicity screening (trypan blue exclusion assay) of a library of 87 novel synthetic C8-linked benzofused PBD monomeric hybrids was carried out using the multiple myeloma cell line, JJN3. Three lead compounds were select- ed for further investigation based on their cytotoxic effects at nanomolar concentrations. The chemical structures of all three compounds, together with that of anthramycin on which they are based, are shown in Figure 1.
In vitro and in vivo cytotoxicity of the lead pyrrolo[2,1-c][1,4]benzodiazepine compounds in multiple myeloma cell lines
The relative cytotoxicity of the three lead compounds was then assessed in five different multiple myeloma cell lines, JJN3, U266, OPM2, MM.1S and H929 using an annexin V/propidium iodide apoptosis assay. The cells were cultured for 48 h in increasing concentrations (1 nM- 100 nM) of DC-1-92, DC-1-170 and DC-1-192 and were compared with untreated controls. Each compound showed a dose-dependent increase in apoptosis; a repre- sentative example of the data generated is shown in Figure 2A. The dose-response curves for each compound were compared in each cell line using overlaid sigmoidal
experiments. In order to investigate the anti-tumor effects of DC-1-192 in vivo, we employed a systemic model of multiple myeloma in which NOD/SCID mice (2 groups of 7 mice) were inoculated with the human RPMI 8226 myeloma cell line (1x107 cells). Treatment was initiated 5 days after inocuation with either DC-1-192 (1 mg/kg) or vehicle control. DC-1-192 was administered once per day (5 days/week) for 3 weeks by intravenous injection and animals were monitored daily for morbidity and mortali- ty. DC-1-192 significantly prolonged the survival of the mice; the median survival of the DC-1-192-treated mice was 68 days versus 56 days in untreated mice (hazard ratio [HR]=2.98; P=0.017) (Figure 2E).
Comparative cytotoxicity in primary chronic lymphocytic leukemia and normal B- and T-lymphocytes Primary CLL cells and age-matched normal B- and T- lymphocytes obtained from healthy donors were treated with increasing concentrations of DC-1-92, DC-1-170 and DC-1-192. Apoptosis was measured using CD19/CD3/annexin V labeling to determine the percent- age of apoptosis induced by the PBD compunds in CD19+ B cells and CD3+ T cells, as shown in Online Supplementary Figure S1A. Online Supplementary Figure S1B shows the comparative dose-responses for each of the cell types indicating that normal lymphocytes were less susceptible to the effects of the PBD. As was the case with the three multiple myeloma cell lines, DC-1-192 was the most potent cytotoxic agent in primary CLL cells. Online Supplementary Figure S1C, D shows that CLL cells were significantly more sensitive to the effects of the PBD than
were age-matched normal B- and T-lymphocytes.
DC-1-192 shows preferential cytotoxicity in chronic lymphocytic leukemia cells carrying a NOTCH1 or BIRC3 mutation
All of the CLL samples treated with DC-1-192 (n=46) showed nanomolar LD50 values with a mean LD50 value for the entire CLL cohort of 3.8 nM (Figure 3A). We next examined whether sensitivity to DC-1-192 was associat- ed with any of the known prognostic markers. There was no significant difference in mean LD50 value between IGHV-mutated and IGHV-unmutated samples (Figure 3B); CD38-positive and CD38-negative samples (≥/<20%) (Figure 3C) and samples with higher or lower
values were then cal- culated for each treatment and plotted on the bar chart shown in Figure 2C. Although each cell line showed dif- ferential sensitivity to the three compounds, in every case DC-1-192 was the most cytotoxic PBD with DC-1-170
plots (Figure 2B) and the mean LD 50
val- ues for DC-1-192 were compared with the published NF- κB index value for each cell line.23 The NF-κB index is the
showing the least cytotoxicity (Figure 2D). The LD
50
values for ten NF-κB-regulated genes (excluding BIRC3/cIAP2); the higher the index value, the more NF-κB-dependent the cell line is deemed to be. With the exception of JJN3 cells, sensitivity to DC-1-192 appeared to be inversely associated with the NF-κB index, a concept we went on to explore in subsequent
We have previously shown that PBD monomers, such as KMR-28-39, have NF-κB inhibitory effects.21 We, there- fore, determined the NF-κB inhibitory properties of this new series of compounds in two myeloma cell lines JJN3 and U266. JJN3 cells overexpress both the canonical and non-canonical NF-κB subunits and possess an EFTUD2- NIK fusion gene which lacks the TRAF3 binding domain resulting in the accumulation of a cytoplasmic EFTUD2-
average of the log
2
-microglobulin concentrations (≥/<3.5 mg/L) (Figure 3D). However, samples derived from patients with a BIRC3 (n=3) or NOTCH1 (n=11) mutation were signifi- cantly more sensitive to the effects of DC-1-192 (Figure 3E) suggesting that elevated NF-κB signaling may be a determinant of sensitivity.24,25 In keeping with this con- cept, the nuclear expression of the NF-κB subunit p65
β
2
(RelA) was inversely correlated with DC-1-192 LD 50
ues (Figure 3F).
Nuclear localization of NF-κB subunits following treatment with pyrrolo[2,1-c][1,4]benzodiazepines
val-
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haematologica | 2021; 106(4)