Myelodysplastic syndromes (MDS) ultimately will progress to a higher-risk form or acute myeloid leukemia. This evolution is accompanied by acquisition of genetic hits following ancestral mutations, ...with further subclonal diversification of the clonal architecture. Conceptually, therapeutic approaches targeting ancestral hits have the potential to eradicate MDS at early stages of ontogenesis. Founder SF3B1 mutations are frequent in MDS and therefore represent rational targets for drug development. During our drug discovery efforts we identified an existing drug that selectively inhibits the growth of SF3B1 mutant (SF3B1MT) cells. We consequently examined the effects of known compounds possibly arresting clonal expansion of SF3B1MT MDS cells.
For our studies, we generated CRISPR/Cas9 knock-in human cells stably expressing the recurrent SF3B1 K700E mutation and a chromophore-tagged reporter (mRFP). Because of the high frequency of SF3B1 mutations and their effects on the splicing of a multitude of genes, we hypothesized that some of the corresponding downstream effects could be effectively targeted. Using luminescent cell viability assays and flow cytometry analysis, we screened a 3,000 compound library for selective sensitivity against isogenic SF3B1MT cells. A subset of this library contained 23 calcium2+ channel blockers (CCBs) of which one specific dihydropirimidine (DHP) showed the highest inhibitory activity against SF3B1MT cells. Several studies point towards a role of calcium signaling in MDS. Proteins active in calcium metabolism (GPR68, calpain, calpastatin) are involved in modulating sensitivity to drugs used in MDS (e.g., lenalidomide). Divalent substrates including Fe2+ may use CCs and it's plausible that our DHP can inhibit iron overload by blocking Fe2+ trafficking through L-type CCs. In this regard, CCBs have been shown to stimulate the activity of adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporters. Furthermore, it is known that the SF3B1 mutations reduce the expression of the iron transporter ABCB7, leading to increased iron accumulation. All of this led us to further evaluate the effects of DHP in vitro and in vivo. DHP had higher growth inhibitory activity against SF3B1MT cells when tested in vitro using 8 serial concentrations in half-log dilutions for a period of 3 days (20% and 60% growth inhibition at 1 and 3μM). Mixed competitive experiment of K700EmRFP and WTGFP cells treated with increased doses of DHP (1, 3, 5, 10, 20, 50 μM) reduced the competitiveness of K700EmRFP over the time inducing a 3-fold reduction at 50μM. K700EmRFP cells expressed half the amount of ABCB7 mRNA compared to WTGFP cells by RT-PCR. Therapeutic index provided by DHP was determined in vivo. Bone marrow cells of B6. Gt(ROSA)26Sortm1Sor/J (CD45.2) donors were transplanted in pre-lethally irradiated B6.SJL-PtprcaPepcb/BoyJ (CD45.1) recipients. Two weeks after transplantation, average engraftment (measured as percentage of CD45.2(+) donor cells) was 96% ± 0.02. DHP (10 mg/Kg) was orally administered. No decrease in the proportion of CD45.2(+) donor cells was seen post-treatment compared to pre-treatment (96±0.01 % vs. 96%±0.009). Similarly no change in the proportion of CD45.2(+) donor cells was observed in competitive repopulation experiments when B6. Gt(ROSA)26Sortm1Sor/J cells were mixed 1:1 with transgenic Sf3b1+/K700E cells (40%±0.13 vs. 44%±0.02). In contrast, preliminary experiments showed that DHP had an effect on reducing Sf3b1+/K700E allele burden in two chimeric mice (to 21% and 24%) compared to pre-treatment. DHP is structurally unique in comparison to other DHP-based CCBs; in that it possess a -CH20-CH2CH2NH2 moiety linked directly to the DHP scaffold that may in itself provide opportunities or modes for Fe2+ chelation as well as its L-type CCB activity. Our observations and structural analyses therefore provide impetus to explore this feature to possibly improve the drug's efficacy.
In sum, we have demonstrated that the clonal growth of cells carrying SF3B1 mutations might be suppressed using the known L-type CCB DHP. This might represent a novel modulator of ATPase activity of ABC transporters in SF3B1MT expressing low ABCB7 levels.
Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.
Proteasomes degrade intracellular proteins to generate antigenic peptides that are recognized by the adaptive immune system and promote anticancer immunity. However, tumors subvert the antigen ...presentation machinery to escape immunosurveillance. We hypothesized that proteasome activation could concomitantly increase antigen abundance and diversity in multiple myeloma (MM) cells. High-throughput screens revealed that histone deacetylase 6 (HDAC6) inhibitors activated proteasomes to unmask neoantigens and amplify the tumor-specific antigenic landscape. Treatment of patient CD138+ cells with HDAC6 inhibitors significantly promoted the anti-myeloma activity of autologous CD8+ T-cells. Pharmacologic blockade and genetic ablation of the HDAC6 ubiquitin-binding domain released HR23B, which shuttles ubiquitinylated cargo to proteasomes, while silencing HDAC6 or HR23B in MM cells abolished the effect of HDAC6 inhibitors on proteasomes, antigen presentation and T-cell cytotoxicity. Taken together, our results demonstrate the paradigm-shifting translational impact of proteasome activators to expand the myeloma immunopeptidome and have revealed novel, actionable antigenic targets for T-cell-directed immunotherapy.
Pairing between the hexamer seed region of a small interfering RNA (siRNA) guide strand (nucleotides 2-7) and complementary sequences in the 3' UTR of mature transcripts has been implicated as an ...important element in off-target gene regulation and false positive phenotypes. To better understand the association between seed sequences and off-target profiles we performed an analysis of all possible (4096) hexamers and identified a nonuniform distribution of hexamer frequencies across the 3' UTR transcriptome. Subsequent microarray analysis of cells transfected with siRNAs having seeds with low, medium, or high seed complement frequencies (SCFs) revealed that duplexes with low SCFs generally induced fewer off-targets and off-target phenotypes than molecules with more abundant 3' UTR complements. These findings provide the first experimentally validated strategy for designing siRNAs with enhanced specificity and allow for more accurate interpretation of high throughput screening data generated with existing siRNA/shRNA collections.
Hypoxia-inducible factor-1 (HIF-1) and its most important subunit, HIF-1alpha, plays a central role in tumor progression by regulating genes involved in cancer cell survival, proliferation and ...metastasis. HIF-1alpha activity is associated with nuclear accumulation of the transcription factor and regulated by several mechanisms including modulation of protein stability and degradation. Among recent advances are the discoveries that inflammation-induced cytokines and growth factors affect protein accumulation of HIF-1alpha under normoxia conditions. TNFalpha, a major pro-inflammatory cytokine that promotes tumorigenesis is known as a stimulator of HIF-1alpha activity. To improve our understanding of TNFalpha-mediated regulation of HIF-1alpha nuclear accumulation we screened a kinase-specific siRNA library using a cell imaging-based HIF-1alpha-eGFP chimera reporter assay. Interestingly, this systematic analysis determined that depletion of kinases involved in conventional TNFalpha signaling (IKK/NFkappaB and JNK pathways) has no detrimental effect on HIF-1alpha accumulation. On the other hand, depletion of PRKAR2B, ADCK2, TRPM7, and TRIB2 significantly decreases the effect of TNFalpha on HIF-1alpha stability in osteosarcoma and prostate cancer cell lines. These newly discovered regulators conveyed their activity through a non-conventional RELB-depended NFkappaB signaling pathway and regulation of superoxide activity. Taken together our data allow us to conclude that TNFalpha uses a distinct and complex signaling mechanism to induce accumulation of HIF-1alpha in cancer cells. In summary, our results illuminate a novel mechanism through which cancer initiation and progression may be promoted by inflammatory cytokines, highlighting new potential avenues for fighting this disease.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
SF3B1 mutations disrupt normal pre-mRNA splicing to cause disease. Drugs inhibiting the interaction between the SF3b complex and RNA or agents degrading auxiliary splicing factors are being tested as ...new avenues for targeted therapy in myeloid neoplasia (MN) with SF3B1 mutations. Here we describe the ability of small molecules to restore altered RNA processes in SF3B1MT MN.
We previously reported (Visconte, ASH 2018) the identification of the small molecule 4-pyridyl-2-anilinothiazole (PAT) which showed growth inhibition of CRISPR/Cas9 SF3B1+/K700E cells and primary SF3B1MT cells. PAT did not influence the growth of other cell models without (THP1, MOLM13FLT3, OCIAML3DNMT3A, SIGM5TET2/DNMT3A, K562PHF6) and with other splicing factor mutations (K562U2AF1, K562LUC7L2). We now describe data from medicinal chemistry, transcriptome, and in vivo studies to advance drug development for SF3B1MT MN. SAR studies focused on logical and systematic modifications of PAT, e.g., i) replacement of the 2,4-disubstituted thiazole spacer ring with other heteroatom containing rings (5,6,7 membered aromatic or aliphatic ring structures); ii) alternative linking groups for the NH linker of the aniline of the tail region (sulfonamide, amide, substituted amine linkers); iii) alternative substituted aromatic and aliphatic ring structures for the phenyl head region substituent, led us to identify permissive sites for further chemical optimization. For example, a 4-chlorophenyl analog demonstrated activity IC50, 3μM similar to PAT. Competitive repopulation assays of bone marrow (BM) cells from dual reporters (ACTBtdTomato; EGFP) B6.GtROSA26 mixed with BM cells from conditional knock-in Sf3b1+/K700E mice injected in pre-lethally irradiated B6.SJL-PtprcaPepcb/BoyJ (CD45.1) recipients (n=18) were used as a preclinical murine model. This model then allowed i) demonstration of drug efficacy in reducing the competitiveness of SF3B1MT cells and ii) evaluation of therapeutic index in normal hematopoiesis. Post-transplant recovery, recipients of B6.GtROSA26 cells underwent PAT treatment (10 mg/Kg/IP/5 days weekly) for a period of 6 weeks without showing any signs of distress or drug intolerance (drop in blood count, weight loss, abdominal swelling, liver or kidney toxicity). Two weeks after transplantation, donor Sf3b1+/K700E cells had an engraftment capability similar to that of donor B6.GtROSA26 cells (83.6 ± 4 vs. 86.4 ± 2.4) when transplanted as a sole graft in CD45.1 recipients. PAT reduced almost half the percentage of Sf3b1+/K700E donor cells at 6 weeks of treatment (47.4%) vs. pre-treatment (83.6%). In mixed (1:1) BM transplants, Sf3b1+/K700E cellshad a repopulative disadvantage against competitors B6.GtROSA26 contributing for 16% of the marrow reconstitution. Similar to single graft transplants, PAT decreased the percentage of Sf3b1+/K700E cells at 6 weeks vs. pre-treatment (average, 6% vs. 16%) in chimeras. Consistent with the lack of toxicity of PAT treatment B6.GtROSA26 cells in chimeras were not affected by PAT and gradually repopulated the host (post-treatment, 80% vs. pre-treatment, 64%). Subsequently, we focused our efforts identifying important genes known to be dysregulated in MDS that were mostly influenced by drug treatment and minimally affected in normal cells. Our approach was based on the analyses of genes linked to erythropoiesis (a key hallmark of low-risk MDS). In normal hematopoiesis TGF-β signaling inhibits terminal erythroid maturation. Out of 13,775 genes, 5% (664/13,775) were found differentially expressed between CRISPR/Cas9 SF3B1+/K700E and parental cells of which 60% of these genes were significantly up-regulated and 40% down-regulated. Pathway analysis showed that the expression levels of SMAD family of genes and GDF factors changed significantly upon drug treatment. SMAD7 mRNA levels are 3-fold lower in MDS CD34+ cells (n=159) compared to the ones of healthy subjects (n=17) (GEO accession GSE58831) leading to TGF-β over activation. PAT treatment normalized SMAD7 expression levels in CRISPR/Cas9 SF3B1+/K700E cells by 3-fold while reducing the levels of GDF11.
In summary, we have identified new drug entities that are modulators of transcriptomic changes which decrease the competitiveness of SF3B1MT cells. These results suggest combination therapies with current TGF-β pathway inhibitors.
Advani:Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy; Amgen: Research Funding; Pfizer: Honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding. Kelly:Novartis, Bayer, Janssen, Pharmacyclics, Celgene, Astrazeneca, Seattle Genetics: Honoraria, Speakers Bureau; Takeda: Research Funding; Genentech, Verastem: Consultancy. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.
Disrupting the formation of the oncogenic YAP/TAZ-TEAD transcriptional complex holds substantial therapeutic potential. However, the three protein interaction interfaces of this complex cannot be ...easily disrupted using small molecules. Here, we report that the pharmacologically active small molecule aurintricarboxylic acid (ATA) acts as a disruptor of the TAZ-TEAD complex. ATA was identified in a high-throughput screen using a TAZ-TEAD AlphaLISA assay that was tailored to identify disruptors of this transcriptional complex. We further used fluorescence polarization assays both to confirm disruption of the TAZ-TEAD complex and to demonstrate that ATA binds to interface 3. We have previously shown that cell-based models that express the oncogenic TAZ-CAMTA1 (TC) fusion protein display enhanced TEAD transcriptional activity because TC functions as an activated form of TAZ. Utilizing cell-based studies and our TC model system, we performed TC/TEAD reporter, RNA-Seq, and qPCR assays and found that ATA inhibits TC/TEAD transcriptional activity. Further, disruption of TC/TEAD and TAZ/TEAD interaction by ATA abrogated anchorage-independent growth, the phenotype most closely linked to dysregulated TAZ/TEAD activity. Therefore, this study demonstrates that ATA is a novel small molecule that has the ability to disrupt the undruggable TAZ-TEAD interface.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
SF3B1 is a splicing factor gene whose mutations are pathognomonic of MDS with ring sideroblasts. Because of the ubiquitous importance of splicing, a major barrier in targeting cells with spliceosomal ...mutations is the discovery of agents decreasing the competitiveness of mutant cells while preserving the integrity of wild type cells. To date no specific therapies are FDA approved for SF3B1 mutant (SF3B1MT) MDS and few agents are in early clinical testing. We describe a novel targeted approach to drug development for SF3B1MT malignancies.
Our investigative strategy started with a high throughput drug screen. We introduced K700E mutation into myeloid cells using CRISPR/Cas9. We then subjected K562+/K700E and matched-parental K562 cells to high throughput drug screen of a library of 3,000 mechanistically annotated, non-redundant, bioactive compounds. Top hits were validated by dose response testing (8 concentrations in half-log dilutions). Our interest focused on compounds with cytostatic activity towards K562+/K700E cells. Among these, a 4-pyridyl-2-anilinothiazole (PAT) showed preferential inhibition of growth in K562+/K700E cells with an IC50 of 3uM. Dose response showed that K562+/K700E cells were significantly sensitive to PAT with a growth inhibition of 20%, 32%, 51%, 65%, 95% at .3uM (P=.01), 1uM (P=.002), 3uM (P<.0001), 10uM (P<.0001), and 20uM (P=.01). High doses (10, 20uM) were toxic to parental cells. PAT treatment did not induce growth arrest in other myeloid cells (THP1, MOLM13FLT3, OCI-AML3DNMT3A, SIG-M5TET2/DNMT3A, K562PHF6) including cells with mutations in other splicing factors (K562U2AF1, K562LUC7L2). PAT induced similar effects in primary SF3B1MT MDS cells at 3uM while it did not induce significant growth inhibition in primary MDS cells with other mutations, including other spliceosomal mutations (e.g.,U2AF1) (N=6). In normal bone marrow cells (N=6), complete growth arrest of erythroid and myeloid cells was observed at high doses (20uM).
Using PAT as our lead, we employed a fragment based reiterative medicinal chemistry approach to synthesize selective compounds and improve therapeutic index. Libraries were constructed following Lipinski rules with ease of synthetic construction in mind. Pilot libraries were constructed via the classic Hantsch thiazole synthesis which involves condensation of α-halo ketones with substituted thioureas. This enterprise investigated SAR modifications of PAT by considering features such as regiochemistry and basicity of the nitrogen of the pyridine ring in the head region; replacement of the spacer 2,4-disubstituted thiazole ring with heteroatom containing rings (5,6,7 membered aromatic or aliphatic ring structures); alternatives for the aniline of the tail region e.g., sulfonamide, amide, and substituted amine linkers and substituted aromatic and aliphatic ring structures for the phenyl substituent. A major challenge in drug development of agents targeting spliceosomal mutations is the identification of key clusters of aberrantly spliced genes restored by drug treatment, e.g., identification of a pharmacodynamic endpoint that could be used to prove that that the drug reached its target. SF3B1 mutations induce aberrant 3′-ss selection by promoting use of alternative branch points. To identify biomarkers of splicing changes to screen our libraries, K562+/K700E and parental cells were treated with PAT (3uM) and RNA libraries were subjected to RNA Seq. The splicing pattern of parental cells was used as reference. We identified 328 cassette exons (±25% splicing inclusion difference, pFDR<.05) in K562+/K700E cells of whom the splicing of 22 exons was partially or completely restored upon treatment. Among these, PAT treatment restored the splicing of exons in sensitive 3′-ss sequences (ENOSF1), RNA binding SR-related factors (ACIN1), zing fingers (ZC3H7A) already associated with SF3B1 downregulation, histone modifiers (HMGN3), mitochondrial genes (TMEM126B), proto-oncogenes (CREB1) and heat shock proteins (DNAJC24). Ongoing experiments include tests of the ability of PATs to restore the splicing of misspliced exons and its efficacy in reducing the percentage of SF3B1MT cells in xenografts of K562+/K700E and primary SF3B1MT cells and Sf3b1+/K700E mice.
In sum, we described novel classes of compounds that inhibit the expansion of SF3B1MT cells by restoring the splicing defects intrinsically associated with SF3B1.
Carraway:Novartis: Speakers Bureau; Jazz: Speakers Bureau; FibroGen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Speakers Bureau. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.
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