HDAC and Proteasome Inhibitors Synergize to Activate Pro-Apoptotic Factors in Synovial Sarcoma

<div><p>Conventional cytotoxic therapies for synovial sarcoma provide limited benefit, and no drugs specifically targeting its driving SS18-SSX fusion oncoprotein are currently available. Patients remain at high risk for early and late metastasis. A high-throughput drug screen consisting of over 900 tool compounds and epigenetic modifiers, representing over 100 drug classes, was undertaken in a panel of synovial sarcoma cell lines to uncover novel sensitizing agents and targetable pathways. Top scoring drug categories were found to be HDAC inhibitors and proteasomal targeting agents. We find that the HDAC inhibitor quisinostat disrupts the SS18-SSX driving protein complex, thereby reestablishing expression of <i>EGR1</i> and <i>CDKN2A</i> tumor suppressors. In combination with proteasome inhibition, HDAC inhibitors synergize to decrease cell viability and elicit apoptosis. Quisinostat inhibits aggresome formation in response to proteasome inhibition, and combination treatment leads to elevated endoplasmic reticulum stress, activation of pro-apoptotic effector proteins BIM and BIK, phosphorylation of BCL-2, increased levels of reactive oxygen species, and suppression of tumor growth in a murine model of synovial sarcoma. This study identifies and provides mechanistic support for a particular susceptibility of synovial sarcoma to the combination of quisinostat and proteasome inhibition.</p></div

Unsuspected osteochondroma-like outgrowths in the cranial base of Hereditary Multiple Exostoses patients and modeling and treatment with a BMP antagonist in mice

<div><p>Hereditary Multiple Exostoses (HME) is a rare pediatric disorder caused by loss-of-function mutations in the genes encoding the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. HME is characterized by formation of cartilaginous outgrowths—called osteochondromas- next to the growth plates of many axial and appendicular skeletal elements. Surprisingly, it is not known whether such tumors also form in endochondral elements of the craniofacial skeleton. Here, we carried out a retrospective analysis of cervical spine MRI and CT scans from 50 consecutive HME patients that included cranial skeletal images. Interestingly, nearly half of the patients displayed moderate defects or osteochondroma-like outgrowths in the cranial base and specifically in the clivus. In good correlation, osteochondromas developed in the cranial base of mutant <i>Ext1</i>^<i>f/f</i><i>;Col2-CreER</i> or <i>Ext1</i>^<i>f/f</i><i>;Aggrecan-CreER</i> mouse models of HME along the synchondrosis growth plates. Osteochondroma formation was preceded by phenotypic alteration of cells at the chondro-perichondrial boundary and was accompanied by ectopic expression of major cartilage matrix genes -<i>collagen 2</i> and <i>collagen X-</i> within the growing ectopic masses. Because chondrogenesis requires bone morphogenetic protein (BMP) signaling, we asked whether osteochondroma formation could be blocked by a BMP signaling antagonist. Systemic administration with LDN-193189 effectively inhibited osteochondroma growth in conditional <i>Ext1</i>-mutant mice. In vitro studies with mouse embryo chondrogenic cells clarified the mechanisms of LDN-193189 action that turned out to include decreases in canonical BMP signaling pSMAD1/5/8 effectors but interestingly, concurrent increases in such anti-chondrogenic mechanisms as pERK1/2 and <i>Chordin</i>, <i>Fgf9</i> and <i>Fgf18</i> expression. Our study is the first to reveal that the cranial base can be affected in patients with HME and that osteochondroma formation is amenable to therapeutic drug intervention.</p></div

HDAC and proteasome inhibition leads to apoptosis via pro-apoptosis protein activation, ROS production and caspase activation.

<p>(A) Pro-apoptotic proteins BIM and BIK are upregulated by both quisinostat and bortezomib, and the drug combination elicits phosphorylation of anti-apoptotic protein BCL-2 in SYO-1 cells. (B) Cleavage of caspase 3 occurs following treatment with the drug combination in synovial sarcoma cell lines, demonstrated by staining with IncuCyte™ Kinetic Caspase-3/7 Apoptosis Assay Reagent, (C) inducing significant apoptosis as confirmed by Annexin-V/PI staining in the SYO-1 cell line (Q3: live, Q2: necrotic/late apoptotic, Q4: early apoptotic). (D) The low-dose quisinostat/bortezomib drug combination brings about a significant decrease in the viability of primary synovial sarcoma cells (83-SS) as compared to matched normal muscle cells derived from the same patient (83-muscle). Two-way ANOVA indicated a significant interaction between cell type and response to the drug combination (<i>p</i> < 0.05). (E) Tumor growth in a murine model of synovial sarcoma was significantly reduced by day 21 with the quisinostat/bortezomib combination treatment, as compared to the vehicle only control. (F) Taken together, the combination of HDAC and proteasome inhibitors results in dissociation of the SS18-SSX driving complex as well as aggresome inhibition, ER stress and ROS production, leading to apoptosis induction in synovial sarcoma. Statistical significance compared to vehicle treatment controls was determined by Student t test or two-way ANOVA where indicated: * denotes <i>p</i> < 0.05. Error bars represent standard error of mean from conditions performed in triplicate. Vinculin was used as a loading control for protein analysis.</p

The synergistic effect of HDAC and proteasome inhibition is consistent within each drug class.

<p>Additional compounds of each drug class were tested in combinational synergy studies. Quisinostat, panobinostat (pan-HDAC inhibitors), and bortezomib, carfilzomib and ixazomib (proteasome inhibitors) were studied in all combinations in the SYO-1 (A) and MoJo (B) SS18-SSX containing cell lines. CI values are less than 1 in these synovial sarcoma cell lines. CI values were calculated using the Chou-Talalay-designed program CompuSyn. Error bars represent standard error of mean from conditions performed in triplicate.</p

HDAC inhibition by quisinostat synergizes with proteasome inhibition to decrease synovial sarcoma cell viability.

<p>(A) In all synovial sarcoma cell lines, but not HEK293T controls, the addition of 0.005 μM of bortezomib results in a downshift of approximately a full log of quisinostat, decreasing the amount of drug required to achieve the same effect as the HDAC inhibitor alone. (B) Isobologram analysis demonstrates synergy of these drug classes in synovial sarcoma cell lines (but not HEK293T controls), as increasing concentration combinations fall below the additive isoboles. (C) Combination index (CI) values calculated for the combination of bortezomib and quisinostat in synovial sarcoma are significantly less than 1, indicating synergy of the compounds is occurring in all six synovial sarcoma cell lines (but not HEK293T controls). Isobolograms and CI values were calculated using the Chou-Talalay-designed program CompuSyn. Statistical significance compared to vehicle treatment controls was determined by Student t test: * denotes <i>p</i> < 0.05; ** denotes <i>p</i> < 0.01; *** denotes <i>p</i> < 0.001. Error bars represent standard error of mean from conditions performed in triplicate. </p

Stereotypic osteochondromas form in a juvenile HME mouse model.

<p>(<b>A-F</b>) Bright field and fluorescence images of longitudinal sections of spheno-occipital (<i>sos</i>) synchondroses (A-C), tibia (D-E) and ribs (F) from 5 week-old control <i>R26-tdTomato;Agr-CreER</i> mice that had been injected with vehicle 3 days earlier. The fluorescence images (B and E) show absence of reporter activity in growth plates (<i>gp</i>) and perichondrium (<i>pc</i>) at any anatomical site examined and thus, lack of <i>CreER</i> leakage in this model. Note that ribs contain a large resting cartilage (<i>rc</i>) adjacent to the growth plate (F). (<b>G-H</b>) μCT images of cranial base and knee from control <i>Ext1</i>^<i>f/f</i><i>;Agr-CreER</i> mice sacrificed 6 weeks from vehicle injection and displaying normal and typical anatomical characteristics. (<b>I-N</b>) Bright field and fluorescence images from companion <i>R26-tdTomato;Agr-CreER</i> mice that were administered tamoxifen once and were sacrificed 3 days post-injection. Note that reporter activity is very strong in spheno-occipital synchondrosis (<i>sos</i>), tibia and rib growth plates and rib resting cartilage (J, K, M and N) and is also conspicuous in flanking perichondrial (<i>pc</i>) cells at each anatomical location (arrowheads in K and M). Boxed area in J is shown at higher magnification in K. (<b>O-P</b>) μCT images of cranial base and knee from mutant <i>Ext1</i>^<i>f/f</i><i>;Agr-CreER</i> mice sacrificed 6 weeks after tamoxifen injection. Note the large multiple osteochondromas (arrowheads) protruding away from the bone surfaces at each anatomical site and better appreciable when contrasted to corresponding images from companion controls (G-H). Bar in (A) for A, B, I and J: 300 μm; bar in (C for C and K, 150 μm; bar in (D) for D, E, L and M, 250 μm; bar in (F) for F and N, 150 μm; and bar in (G) for G and O, 0.8 mm; and in (H) for H and P, 5 mm.</p

Osteochondroma development in long bones and ribs in juvenile mice is inhibited by systemic LDN-193189 treatment.

<p><b>(A to D)</b> μCT images (A, B and D) and X-ray image (C) of knee and ribs from mutant <i>Ext1</i>^<i>f/f</i><i>;Agr-CreER</i> mice receiving vehicle treatment and showing large osteochondromas (double arrowheads). (<b>E to H</b>) Analogous images from companion mutant mice receiving LDN treatment for 6 weeks. Note the appreciable reduction in osteochondorma size in both knee and ribs (arrowhead). Bar in (A) for A, B, E and F, 5 mm; bar in (C) for C and G, 3 mm; and bar in (D) for D and H, 1.5 mm.</p

High-throughput drug screen reveals HDAC and proteasome inhibitors as potent drug classes against synovial sarcoma.

<p>(A) Compounds resulting in measured relative cell viability of less than 50% are annotated as hits (blue). Y-axis denotes drug target classes arranged in alphabetical order. (B) The top 40 drug screen hits out of the 900 compound screen represented by drug target class, demonstrate HDAC inhibitors as the most abundant hits in the screen. (C) Compounds that brought about greater than 90% decreased relative cell viability were scored as 1 (+++), 75.1–90% as 0.5 (++), 50–75% as 0.25 (+) and less than 50% as 0 (-). Total score across the six cell lines was calculated out of 6. (D) IC₅₀ measurements were calculated for drug screen hits quisinostat (HDAC inhibitor), BGT-226 (PI3K/mTOR inhibitor), bortezomib (proteasome inhibitor) as compared with the current standard for synovial sarcoma treatment doxorubicin (cytotoxic DNA/RNA intercalating agent and topoisomerase inhibitor), in a panel of six human SS18-SSX positive cell lines and two control cell lines (HEK293T, MCF7). Error bars signify standard error of mean from conditions performed in triplicate.</p

Cranial base osteochondromas display growth plate-like gene expression patterns.

<p>(<b>A-E</b>) Longitudinal serial sections through the spheno-occipital synchondrosis and flanking tissues from control mice at one month from tamoxifen injection. Sections were stained with safranin O-fast green to reveal cartilage organization and intact perichondrium (<i>pc</i>) and border (arrows) (A), and were processed for in situ hybridization expression analysis of such typical growth plate genes as: (B) collagen II (<i>Col II</i>); (C) collagen X (<i>Col X</i>); (D) metalloprotease 13 (<i>Mmp13</i>); and (E) collagen I (<i>Col I</i>). Arrowheads in B-D point to characteristic areas/sites of maximal gene expression in growth plates and arrows in E point to <i>Col I</i> expression in bone collar. (<b>F-J</b>) Longitudinal serial sections through osteochondromas present along the nasal aspect of cranial base in companion mutant tamoxifen-injected <i>Ext1</i>^<i>f/f</i><i>;Col2-CreER</i> mice. Arrows in F point to osteochondromas protruding away from the cranial base into surrounding perichondrium and nasal cavity. Arrowheads in G-I point to areas of maximal and somewhat deranged gene expression patterns within the osteochondromas. Arrows in J point to <i>Col I</i> expression in bone collar. Bar in (F) for all panels, 200 μm.</p

Osteochondromas form in the cranial base of <i>Ext1</i> mutant mice over time.

<p>(<b>A-F</b>) Longitudinal sections through the spheno-occipital synchondrosis (<i>sos</i>) in control tamoxifen-injected mice at 2 weeks (A-C), 1 month (D), 3 months (E) and 6 months (F) from injection stained with safranin O and fast green. Note in (B) the characteristic growth plate organization with resting (<i>rt</i>), proliferative (<i>pl</i>), prehypertrophic (<i>ph</i>) and hypertrophic (<i>hp</i>) zones, and note in (C) the distinct inner and outer portions of perichondrium (<i>pc</i>) (arrow and double arrowhead, respectively) along the perichondrium/growth plate (PC/GP) border. (<b>G-R</b>) Longitudinal sections through the spheno-occipital synchondrosis in <i>Ext1</i>^<i>f/f</i><i>;Col2-CreER</i> mutant mice at 2 weeks (G-I), 3 weeks (M-O), 1 month (J, P), 3 months (K, Q) and 6 months (L, R) from tamoxifen injection. Note that the chondro-perichondrial border is already deranged at 2 weeks as indicated by presence of round, safranin O-positive cells within perichondrium (I, arrowhead). By 3 weeks and thereafter, the border becomes occupied by incipient osteochondromas (O, arrowheads) that enlarge (J) and ossify proximally over time (K and L, asterisks) and maintain a characteristic cartilage cap at their distal end (K and L, arrowheads) with a growth plate-like organization (P and R) with scattered Edu-labeled proliferative cells (Q, arrrowheads). Bar in (A) for A, G and M, 1 mm; bar in (C) for B, C, H, I, N and O, 50 μm; bar in (D) for D, E, F, J-L and R, 3 mm; bar in (P) for P and Q, 300 μm.</p