Identification of KIT as a crucial oncogenic regulator pathway has revolutionized the treatment of GIST(25
). The KIT-inhibitor imatinib has tripled the median survival of patients with metastatic GIST and many patients live 5 years or longer with the disease. However, most patients inevitably develop resistance. Resistance is mostly conferred by secondary mutations within the split kinase domain of KIT(6
). While resistance mutations within the ATP-binding domain (T670I, V654A) can successfully be targeted by alternative KIT-inhibitors such as sunitinib, mutations within the activation loop are an ongoing pharmacologic challenge (29
). In addition, more than 9 different resistance mutations have already been described and different mutations may even occur within the same patient(6
). Direct, ATP-competitive inhibitors are therefore unlikely to sufficiently inhibit all possible KIT mutations.
Alternative strategies aim to inhibit the oncogenic signal of KIT regardless of existing secondary mutations. Among those are inhibition of KIT-dependent signaling pathways (e.g. PI3K, AKT or mTOR(34
)) or indirect inhibition of KIT using HSP90 inhibitors(9
) which are already being tested clinically.
The post-translational modification of histones, e.g. through acetylation and deacetylation of lysine-tails, has been shown to be an important mechanism of transcriptional regulation(38
). Interestingly, many genes upregulated by HATs are important for differentiation, cell cycle control and apoptosis(39
). Aberrant acetylation, either through overexpression of HDAC or HAT dysfunction is commonly found in epithelial and hematological cancers (40
). In this context HDAC inhibitors exhibit an apparent selectivity for activating transcription of tumor-suppressing genes(39
Recently, several groups have highlighted the potential therapeutic relevance of HDAC inhibitors in IM-resistant chronic myelogenous leukemia cells. HDACI destabilized the BCR-ABL oncoprotein even in the presence of the highly IM-resistant T315I gatekeeper mutation(13
). Given the similarities of the oncogenic tyrosine-kinase-mechanisms between CML and GIST we therefore investigated the potential therapeutic value of HDACI in GIST.
The data reported herein are the first to show strong antiproliferative and proapoptotic effects in both IM-sensitive and IM-resistant GIST by inhibitors of histone deacetylases. We show that these effects are conferred by inactivation of the KIT-oncogenic signaling cascade. The inactivation mechanisms include both inhibition of KIT transcript expression and also degradation of KIT oncoproteins via suppression of deacetylation of the KIT chaperone HSP90.
KIT-positive GIST lines were sensitive to HDACI (IC50 1.7–3.5 μM), while KIT-negative GIST lines were unresponsive (). These findings compare well with data from other SAHA sensitive cancer cell lines, for which IC50s (SAHA) in the low micromolar dose range were described(15
). These dose levels are considered to be therapeutically achievable. Antiproliferative effects of HDACI were lower in non-GIST sarcomas, with only 10–20% inhibition after treating leiomyosarcoma (LMS03) and liposarcoma (LPS141) cell lines with 10 μM SAHA indicating that GISTs are uniquely susceptible to HDACI.
In KIT-positive GISTs, HDACI treatment induced dose-dependent inhibition of KIT phosphorylation, paralleled by AKT inhibition (). In GIST882, inhibition of AKT-phosphorylation was even more pronounced with all HDACI used than inhibition of KIT phosphorylation (, Supplemental Figure 1
), suggesting that HDACI may have inhibitory effects on the PI3K-AKT pathway independent of KIT inhibition. Interestingly, in studies with different HDACI, the effects on histone acetylation did not strictly correlate with KIT inhibition, suggesting that KIT-inhibitory effects are partly histone independent and perhaps dependent on certain HDAC subtypes. While TSA, SAHA and LBH589 are pan HDACI, VPA and NaB only inhibit class I HDACs.
Our studies suggest that HDACI have several mechanisms contributing to KIT inhibition: Histone acetylation may cause reduced KIT transcription, most likely by affecting expression of KIT transcriptional repressors. HDACI might also regulate oncoprotein function, e.g. by modifying KIT acetylation, and may influence KIT oncogenic signaling through effects on non-histone HDACI targets.
Direct, ATP-competitive inhibitors of KIT, such as IM, are known to have KIT-inhibitory effects within several minutes of treatment (data not shown). Time course-studies with HDACI () showed onset of KIT inhibition after 3 and 6 hours of treatment, with a maximum effect between 12 and 18 hours. Only subtle histone acetylation was seen at the early time points (3–6h) and the maximal acetylation level was seen only at the last time point (24h). These findings suggest the possibility of HDACI-mediated indirect inhibition of KIT rather than a direct, biochemical inhibition as seen with IM. Notably, signalling studies demonstrated both inhibition of KIT phosphorylation and decreased KIT expression. While direct KIT inhibitors such as IM and sunitinib do not decrease total KIT expression, we have previously shown that HSP90 inhibitors strongly decrease KIT expression levels(9
HSP90 is known to be subject to several posttranslational modifications that affect its function. Hyperacetylation of HSP90, especially acetylation of the amino acid K294, decreases the affinity for most clients and certain cochaperones(46
). Inhibition of HDAC6, a class II HDAC, has recently been shown to disrupt the chaperone function of HSP90 resulting in degradation of HSP90 clients, such as BCR-ABL(12
). This appears to be caused by lower affinity for ATP(43
). As the assembly of the functional HSP90 chaperone complex requires ATP, HDACI effects on HSP90 can be explained in part by the inhibition of its complex formation. In line with this model we here show that HDACI treatment of GIST cells causes dissociation of HDAC6 from HSP90 resulting in hyperacetylation of HSP90 and a consequent loss of KIT ().
Treatment of GIST48B and GIST62 with SAHA resulted in substantial inhibition of pMAPK (GIST48B and GIST62) and pAKT (GIST62) despite the lack of measurable KIT activation (, data not shown). These findings indicate that apart from HDACI effects on KIT, other signaling pathways might be affected.
Of note, KIT
transcript levels were found to be downregulated in a time- and dose-dependent fashion, with maximal effects seen after 12 hours of SAHA treatment (). Since HDACI are thought to enable rather than suppress transcription, these effects can be explained by induction of a gene responsible for KIT
transcriptional repression. The loss of KIT expression seen after 12 hours of treatment with HDACI could be explained both by transcriptional repression and HSP90 inhibition. However, the extent of KIT inhibition due to a transcriptional block at this time point would mainly depend on the half-life of mature KIT in GIST cells. We therefore treated GIST882 with cycloheximide and demonstrated that the KIT half-life, in GIST, is less than 3 hours (). In contrast, the HSP90 chaperone (>24h) or the KIT regulator PKCtheta (>12h) exhibited a more than four times longer half life following cycloheximide treatment (). Ou et al. recently reported that PKCtheta is a major regulator of KIT transcription(24
). Notably, HDACI caused partial inhibition of PKCtheta, and PKCtheta inhibition may therefore contribute to the inhibition of KIT expression in GIST.
Mechanisms of resistance to direct KIT-inhibitors largely depend on secondary mutations within the KIT ATP binding pocket or activation loop. Our studies suggest that HDACI-mediated inhibition of KIT oncoproteins is independent of the KIT mutation location, with equal relevance for GISTs with imatinib-sensitive or imatinib-resistant KIT mutations. Hence, HDACI might overcome resistance to both ATP binding pocket and activation loop imatinib-resistance mutations, providing a new strategy to inhibit KIT.
Similar to studies evaluating combinations of IM and HDACI in CML, we found no evidence for antagonistic effects and we identified additive proapoptotic effects in GIST882 and GIST48(15
). Notably, we observed synergistic effects of HDACI and IM on histone acetylation in the KIT-positive GIST lines () but not in the KIT-negative GIST48B.
We observed induction of p21 by SAHA in all GIST cell lines (), including KIT-negative GIST48B and GIST62 (, data not shown), and this might result directly from histone acetylation (49
). However, in the KIT-negative GIST48B and GIST62, p21 induction did not cause marked cell cycle arrest, nor induction of apoptosis (, Supplemental Figures 3
; data not shown). These findings suggest that inhibition of oncogenic KIT is the most consequential mechanism of HDACI action in GIST.
Taken together, our data show that HDACI have disease specific effects in GIST by inhibiting the crucial KIT oncogenic pathway. We consistently found additive effects of HDACI and IM in IM-sensitive GIST, regardless of the HDACI used. LBH589 exhibits the highest potency of all HDACI tested even though biological effects of SAHA and LBH589 are similar. Possible mechanisms of action for HDACI in GIST include acetylation of HSP90 with consequent destabilization of KIT, but effects on KIT transcriptional activity may also play a partial role. Given the antiproliferative and proapoptotic effects of HDACI at doses that can be achieved therapeutically, our data provide a strong rationale for the clinical evaluation of HDACI/DACI in GIST.