Voruciclib, a clinical stage oral CDK9 inhibitor, represses MCL-1 and sensitizes high-risk Diffuse Large B-cell Lymphoma to BCL2 inhibition

Abstract

Aberrant regulation of BCL-2 family members enables evasion of apoptosis and tumor resistance to chemotherapy. BCL-2 and functionally redundant counterpart, MCL-1, are frequently over-expressed in high-risk diffuse large B-cell lymphoma (DLBCL). While clinical inhibition of BCL-2 has been achieved with the BH3 mimetic venetoclax, anti-tumor efficacy is limited by compensatory induction of MCL-1. Voruciclib, an orally bioavailable clinical stage CDK-selective inhibitor, potently blocks CDK9, the transcriptional regulator of MCL-1. Here, we demonstrate that voruciclib represses MCL-1 protein expression in preclinical models of DLBCL. When combined with venetoclax in vivo, voruciclib leads to model-dependent tumor cell apoptosis and tumor growth inhibition. Strongest responses were observed in two models representing high-risk activated B-cell (ABC) DLBCL, while no response was observed in a third ABC model, and intermediate responses were observed in two models of germinal center B-cell like (GCB) DLBCL. Given the range of responses, we show that CIVO, a multiplexed tumor micro-dosing technology, represents a viable functional precision medicine approach for differentiating responders from non-responders to BCL-2/MCL-1 targeted therapy. These findings suggest that the combination of voruciclib and venetoclax holds promise as a novel, exclusively oral combination therapy for a subset of high-risk DLBCL patients.

Figure 1

Comparative kinase profiles of voruciclib and flavopiridol. (a) Notable differences in the structures of voruciclib and flavopiridol - First, in the C ring, voruciclib contains a trifluoromethyl (CF3) group at the 4-position of the phenyl ring (shown in red). Second, the D rings of the two molecules contain different saturated heterocycles, each with a basic nitrogen and a hydroxyl or hydroxymethyl substituent: 2-hydroxymethyl-1-methylpyrrolidin-3-yl (voruciclib) and 3-hydroxy-1-methylpiperidiny-4-yl (flavopiridol). Third, the hydroxyl group of voruciclib is connected to C8 by 3 carbons whereas that of flavopiridol is connected by 2 carbons. (b) Kinase dendograms for voruciclib and flavopiridol - Of the 48 consensus hits from the DiscoverRx and Thermofisher kinase screens that were followed up for functional IC50 determination (Reaction Biology), the kinases which bound at K_i values under 3000 nM for voruciclib and flavopiridol respectively are shown in the dendograms with red circles, where larger circles indicate higher-affinity binding. (c,d) Comparison of K_i values for voruciclib and flavopiridol targets shows CDK targets (green) and non-CDK targets (black). High affinity non-CDK targets of flavopiridol (ICK and MAK) are shown in red.

Figure 2

Voruciclib at clinically relevant concentrations leads to down regulation of MCL-1 protein (a) Predicted plasma concentrations in humans dosed daily orally with 250 mg voruciclib. Maximum, average and minimum plasma levels are shown by the red, green and blue lines respectively. The parameter set derived from the PK data after 13/15 days dosing was used for the 15-day simulation for the steady state levels attained after repeat dosing. (b) Western blot analyses for RNA POL II (pSer2), MCL-1, cPARP using protein lysates isolated from SU-DHL-4 cells. β actin was used as the loading control. (c) Western blot analyses for MCL-1 and cPARP from protein lysates isolated from U2932, RIVA, OCI-LY10 cells (ABC subtype) and NU-DHL-1, SU-DHL-4 and SU-DHL-6 cells (GCB subtype) treated with voruciclib (0.5 µM–5 µM) for 6 hours. β actin was used as the loading control. (d,e) Western blot and immunohistochemical (IHC) analyses for MCL-1, using tumor lysates and formalin-fixed paraffin-embedded sections respectively, obtained from OCI-LY10 xenograft bearing mice (n = 3 per condition) orally treated with vehicle, voruciclib 100 mpk or 200 mpk for 5 consecutive days. Tumors were resected 4 hours after the final dose and processed for both Western blot and IHC analyses. β actin was used as loading control for the Western blot where each lane represents an individual tumor (Tumor (T) #1-9). MCL-1 signals, normalized to β actin, were quantified and the average MCL-1 signal for each condition relative to vehicle is shown in the corresponding bar graph. Error bars represent standard error of the mean (SEM). Asterisks (*) denote p value < 0.05 calculated using the Student’s t-test for comparison of vehicle vs treatment. DAPI was used as counterstain for IHC. Representative images are shown. Scale bar: 1000 µm.

Figure 3

Voruciclib combined with venetoclax leads to model-dependent tumor growth inhibition and long term systemic benefits. (a) Tumor growth plots from U2932, RIVA, OCI-LY10, NU-DHL-1 and SU-DHL-4 xenografted mice (n = 5–6 per treatment condition for each model) respectively, orally treated with vehicles corresponding to both drugs (control arm), venetoclax, voruciclib or a combination of both drugs at the same dose and frequency as the corresponding single agents (see materials and methods for dosing regimens). Arrows indicate start and end day of treatment (tx). Drug efficacy with respect to vehicle was assessed in all arms via tumor volume measurements which are averaged across all subjects in the respective cohorts and plotted over time in days. Error bars represent SEM. (b) Kaplan Meier survival plots for each treatment cohort in the U2932 study are shown where xenografted mice were followed for up to 62 days after cessation of treatment. Asterisk (*) denotes p value < 0.05 calculated using the Log-rank test for comparison of combination versus single agent survival curves. (c) Percent tumor growth inhibition (TGI) relative to respective vehicle controls, were estimated using tumor volume measurements at Day 18 (U2932), Day 21 (RIVA), Day 22 (OCI-LY10), Day 18 (NU-DHL-1) and Day 25 (SU-DHL-4) (see methods). p values for TGI comparisons were calculated using the Mann-Whitney test.

Figure 4

: Localized tumor apoptosis following drug microdosing with CIVO differentiates sensitive from resistant ABC DLBCL models, in vivo. (a) For combination analysis, U2932, RIVA, OCI-LY10 tumors (ABC subtype) were micro-injected with vehicle, voruciclib, venetoclax and voruciclib + venetoclax (U2932 n = 7; RIVA n = 12; OCI-LY10 n = 8 tumors) and resected 24 hours post injection. Fluorescent tracking marker (FTM) demarcates the sites of injection. Tissue sections were stained for cleaved caspase 3 (CC3) and DAPI. Representative images are shown. Scale bar: 500 µm (b) Radial effect plots show the difference between fraction CC3 + cells induced by voruciclib, venetoclax or the combination versus the vehicle control, as a function of radial distance from the site of injection demarcated by FTM. Error bars denote 95% confidence intervals. Asterisks (*) indicate statistical significance (p values < 0.05) at radial distances where CC3 responses induced by the combination are greater than both single agents.

Figure 5

BCL-xL, a potential mediator of resistance, is upregulated in response to MCL-1/BCL-2 inhibition (a) Western blot analysis for BCL-xL protein using tumor lysates (previously described in Fig. 2d) derived from OCI-LY10 xenograft bearing mice (n = 3 per treatment) systemically treated with vehicle, voruciclib 100 mpk or 200 mpk by oral gavage for 5 consecutive days followed by tumor resections 4 hours after the final dose. β actin was used as the loading control. Each lane represents an individual tumor (Tumor (T) #1–9). BCL-xl signals, normalized to β actin, were quantified and the average BCL-xl signal for each condition relative to vehicle is shown in the corresponding bar graph. Error bars represent SEM. Asterisks (*) denote p value < 0.05 calculated using the Student’s t-test for comparison of vehicle vs treatment. (b) Western blot analyses for BCL-xL protein using lysates derived from ABC cell lines (OCI-LY10, U2932 and RIVA) treated with vehicle, voruciclib 5 µM (all lines), venetoclax (OCI-LY10 and U2932: 50 nM, RIVA 10 nM) or combination thereof, for 24 hours. β actin was used as the loading control.