Small molecule epigenetic screen identifies novel EZH2 and HDAC inhibitors that target glioblastoma brain tumor-initiating cells

Abstract

Glioblastoma (GBM) is the most lethal and aggressive adult brain tumor, requiring the development of efficacious therapeutics. Towards this goal, we screened five genetically distinct patient-derived brain-tumor initiating cell lines (BTIC) with a unique collection of small molecule epigenetic modulators from the Structural Genomics Consortium (SGC). We identified multiple hits that inhibited the growth of BTICs in vitro, and further evaluated the therapeutic potential of EZH2 and HDAC inhibitors due to the high relevance of these targets for GBM. We found that the novel SAM-competitive EZH2 inhibitor UNC1999 exhibited low micromolar cytotoxicity in vitro on a diverse collection of BTIC lines, synergized with dexamethasone (DEX) and suppressed tumor growth in vivo in combination with DEX. In addition, a unique brain-penetrant class I HDAC inhibitor exhibited cytotoxicity in vitro on a panel of BTIC lines and extended survival in combination with TMZ in an orthotopic BTIC model in vivo. Finally, a combination of EZH2 and HDAC inhibitors demonstrated synergy in vitro by augmenting apoptosis and increasing DNA damage. Our findings identify key epigenetic modulators in GBM that regulate BTIC growth and survival and highlight promising combination therapies.

Keywords: HDAC inhibitor; UNC1999; drug discovery; epigenetics; glioblastoma.

Figure 1. Testing of the SGC epigenetic library of chemical probes on five BTICs lines

A. Detailed design of the screening procedure; B. Epigenetic screen results. Compounds that exhibit IC₅₀ < 5 μM were defined as hits and are shown in red, drugs with 5 μM < IC₅₀ < 10 μM are marked in yellow and inactive compounds that exhibit IC₅₀ > 10 μM are shown in green.

Figure 2. EZH2 is overexpressed in BTICs and SAM-competitive EZH2 inhibitors, in particular UNC1999, are cytotoxic in vitro

A. EZH2 mRNA expression in BTICs. B. EZH2 protein expression in BTICs and SKPs by immunoblotting with anti-EZH2 antibody. C. IC₅₀ values presented as bar graph for SAM-competitive EZH2 inhibitors using alamarBlue as a read-out.

Figure 3. UNC1999 treatment decreases cell viability, impairs self-renewal, causes cell cycle arrest and decreases H3K27Me3

A. Treatment with UNC1999 (4-6 μM) reduces viable cell numbers in BT73 as assessed by trypan blue exclusion (n=3; ** P<0.01). B. Treatment with varying concentrations of UNC1999 impairs self renewal in BT73 and BT147 as assessed by sphere assay (n=3). C. Treatment with UNC1999 (2-5 μM) induces G1 cell cycle arrest in BT73 as assessed by propidium iodide staining. D. Representative western blot demonstrates the effect of treatment with UNC1999 and the negative control UNC2400 on H3K27Me3, total levels of Histone H3, cleaved-PARP and LC3B in BT73 and BT147. E. Treatment with UNC1999 (5 μM) and ZVAD-FMK (20 μM) for 72 hours does not rescue cell viability in BT73 as assessed by trypan blue exclusion (n=3; ** P<0.01). F. Treatment with UNC1999 (5 μM) and Necrostatin (50 μM) for 72 hours does not rescue cell viability in BT73 as assessed by trypan blue exclusion (n=3; ** P<0.01).

Figure 4. UNC1999 is synergistic with dexamethasone (DEX) in vitro and suppresses tumor growth in vivo in a flank xenograft model

A. Representative bar graphs demonstrating synergy between UNC1999, 3.7 μM and DEX, 31 μM in vitro. B. Representative western blots demonstrating assessment of different targets following treatment with drugs alone and a combination in vitro. C. NOD/SCID mice bearing ~25 mm³ tumors were randomized into 4 groups: 1) vehicle; 2) UNC1999, 150 mg/kg, gavage; 3) DEX, 1 mg/kg, i.p. and 4) combination. Three independent experiments were performed. Representative tumor growth data and tumor volume at 2 weeks after treatment initiation are shown (*p<0.05; ** p<0.01).

Figure 5. Treatment with brain-penetrant HDAC inhibitor compound 26 decreases cell viability, impairs self-renewal, causes cell cycle arrest, induces apoptosis and increases total levels of acetylated H3

A. Compound 26 is cytotoxic on diverse BTIC lines in vitro. Waterfall graph with IC₅₀ values is shown. B. Treatment with compound 26 (100-400 nM) reduces viable cell numbers in BT73 as assessed by trypan blue exclusion (n=3; ** P<0.01). C. Treatment with varying concentrations of compound 26 impairs self renewal in BT73 and BT147 as assessed by sphere assay (n=3). D. Treatment with compound 26 (200-1000 nM) induces G1 cell cycle arrest in BT73 as assessed via Propidium iodide staining. E. Representative western blot demonstrates effect of treatment with compound 26 (100-400 nM) on total acetylation of histone H3, total levels of Histone H3, HDAC1/2, p21 and cleaved PARP in BT73 and BT147. F. Treatment with compound 26 (400-1000 nM) increases the percentage of early and late apoptotic cells as assessed via Annexin V staining (n=3).

Figure 6. HDAC inhibitor compound 26 crosses the blood brain barrier and extends survival in an orthotopic tumor model in combination with TMZ

A. Pharmacokinetic results demonstrating plasma and brain concentrations in NOD/SCID mice treated with different doses of compound 26 over 5 days. B. NOD/SCID mice bearing intracranial tumors were randomized into 4 groups: 1) vehicle; 2) compound 26, 10 mg/kg, gavage; 3) TMZ, 30 mg/kg, gavage and 4) combination of the 2 compounds. Kaplan-Meyer survival plot is shown. (* p<0.05; ** p<0.01).

Figure 7. HDAC inhibitor compound 26 synergizes with UNC1999 in vitro through augmented apoptosis and DNA damage

A. Representative bar graphs demonstrating synergy between UNC1999, 3.8 μM and compound 26, 385 nM in vitro (n=3; * p<0.05; ** p<0.01). B. Representative western blots demonstrating assessment of different targets following treatment with drugs alone and a combination in vitro.