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Supplementary Table 4: Genes down-regulated (> 2-fold) by Stat5-shRNA and IST5-002 in K562

Supplementary Methods. Supplementary Figure 1. Chemical structures of the small molecule IST5-002 analogs. Supplementary Figure 2. IST5-002 inhibits phosphorylation of Stat5 in mouse and human breast cancer cells. Supplementary Figure 3. IST5-002 does not have significant inhibitory activity against 50 kinases tested. Supplementary Figure 4. IST5-002 does not affect phosphorylation of Pak1/2. Supplementary Figure 5. IST5-002 inhibits expression of Stat5 target genes in PC cells and in CML cells. Supplementary Figure 6. IST5-002 is not generally cytotoxic as determined by lack of cell death induction in cancer cell lines established from other organs. Supplementary Figure 7. CWR22Rv1 PC cells were inoculated subcutaneously into the flanks of castrated athymic nude mice supplied with sustained-release 5alpha-dihydrotestosterone (DHT)-pellets (n=10/group, 1 tumor/mouse, 1.5 ??107 CWR22Rv1 cells per site, 1 DHT pellet/mouse). Supplementary Figure 8. IST5-0-02 induces apoptosis in CWR22Rv1 tumors grown in nude mice and in clinical PCs cultured ex vivo in organ explant cultures. Supplementary Figure 9. IST5-002 induces extensive apoptotic death of imatinib-sensitive and -resistant CML cells.

Supplementary Table 3: Genes up-regulated (> 2-fold) by Stat5-shRNA and IST5-002 in K562

Supplementary Table 5: IST5-002 regulation of known Stat5 target genes in K562 cells.

Supplementary Table 2: Genes regulated by both Stat5-shRNA and IST5-002 in K562 cells, corresponding to the heatmap in Figure 2g.

Supplemental Table 1: Structure-Activity-Relationship analysis.

Despite treatment with intensive chemotherapy, acute myelogenous leukemia (AML) remains an aggressive malignancy with a dismal outcome in most patients. We found that AML cells exhibit an unusually rapid accumulation of the repressive histone mark H3K27me3 on nascent DNA. In cell lines, primary cells and xenograft mouse models, inhibition of the H3K27 histone methyltransferase EZH2 to decondense the H3K27me3-marked chromatin of AML cells enhanced chromatin accessibility and chemotherapy-induced DNA damage, apoptosis, and leukemia suppression. These effects were further promoted when chromatin decondensation of AML cells was induced upon S-phase entry after release from a transient G1 arrest mediated by CDK4/6 inhibition. In the p53-null KG-1 and THP-1 AML cell lines, EZH2 inhibitor and doxorubicin cotreatment induced transcriptional reprogramming that was, in part, dependent on derepression of H3K27me3-marked gene promoters and led to increased expression of cell death–promoting and growth-inhibitory genes.In conclusion, decondensing H3K27me3-marked chromatin by EZH2 inhibition represents a promising approach to improve the efficacy of DNA-damaging cytotoxic agents in patients with AML. This strategy might allow for a lowering of chemotherapy doses, with a consequent reduction of treatment-related side effects in elderly patients with AML or those with significant comorbidities.Significance:Pharmacological inhibition of EZH2 renders DNA of AML cells more accessible to cytotoxic agents, facilitating leukemia suppression with reduced doses of chemotherapy.See related commentary by Adema and Colla, p. 359

Primary human Ph+ ALL samples

Combining standard cytotoxic chemotherapy with BCR-ABL1 tyrosine kinase inhibitors (TKI) has greatly improved the upfront treatment of patients with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). However, due to the development of drug resistance through both BCR-ABL1–dependent and -independent mechanisms, prognosis remains poor. The STAT5 transcription factor is activated by BCR-ABL1 and by JAK2-dependent cytokine signaling; therefore, inhibiting its activity could address both mechanisms of resistance in Ph+ ALL. We show here that genetic and pharmacologic inhibition of STAT5 activity suppresses cell growth, induces apoptosis, and inhibits leukemogenesis of Ph+ cell lines and patient-derived newly diagnosed and relapsed/TKI-resistant Ph+ ALL cells ex vivo and in mouse models. STAT5 silencing decreased expression of the growth-promoting PIM-1 kinase, the apoptosis inhibitors MCL1 and BCL2, and increased expression of proapoptotic BIM protein. The resulting apoptosis of STAT5-silenced Ph+ BV173 cells was rescued by silencing of BIM or restoration of BCL2 expression. Treatment of Ph+ ALL cells, including samples from relapsed/refractory patients, with the PIM kinase inhibitor AZD1208 and/or the BCL2 family antagonist Sabutoclax markedly suppressed cell growth and leukemogenesis ex vivo and in mice. Together, these studies indicate that targeting STAT5 or STAT5-regulated pathways may provide a new approach for therapy development in Ph+ ALL, especially the relapsed/TKI-resistant disease.Significance:Suppression of STAT5 by BCL2 and PIM kinase inhibitors reduces leukemia burden in mice and constitutes a new potential therapeutic approach against Ph+ ALL, especially in tyrosine kinase inhibitor-resistant disease. Cancer Res; 78(20); 5793–807. ©2018 AACR.

Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ ALL) is currently treated with BCR-ABL1 tyrosine kinase inhibitors (TKI) in combination with chemotherapy. However, most patients develop resistance to TKI through BCR-ABL1–dependent and –independent mechanisms. Newly developed TKI can target Ph+ ALL cells with BCR-ABL1–dependent resistance; however, overcoming BCR-ABL1–independent mechanisms of resistance remains challenging because transcription factors, which are difficult to inhibit, are often involved. We show here that (i) the growth of Ph+ ALL cell lines and primary cells is highly dependent on MYB-mediated transcriptional upregulation of CDK6, cyclin D3, and BCL2, and (ii) restoring their expression in MYB-silenced Ph+ ALL cells rescues their impaired proliferation and survival. Levels of MYB and CDK6 were highly correlated in adult Ph+ ALL (P = 0.00008). Moreover, Ph+ ALL cells exhibited a specific requirement for CDK6 but not CDK4 expression, most likely because, in these cells, CDK6 was predominantly localized in the nucleus, whereas CDK4 was almost exclusively cytoplasmic. Consistent with their essential role in Ph+ ALL, pharmacologic inhibition of CDK6 and BCL2 markedly suppressed proliferation, colony formation, and survival of Ph+ ALL cells ex vivo and in mice. In summary, these findings provide a proof-of-principle, rational strategy to target the MYB "addiction" of Ph+ ALL.Significance: MYB blockade can suppress Philadelphia chromosome-positive leukemia in mice, suggesting that this therapeutic strategy may be useful in patients who develop resistance to imatinib and other TKIs used to treat this disease. Cancer Res; 78(4); 1097–109. ©2017 AACR.

List of primers and antibodies used.

Supplemental Table IIList of Primers used for Real Time PCR experiments

Supplementary DataSupplemental Methods, Supplemental Figures and Legends

Supplementary Fig. S1. Effect of Doxycycline on shScramble (shSCR)-transduced Ph+ ALL cell lines. Supplementary Fig. S2. Effect of STAT5 inhibitor IST5-002 on Ph+ leukemia lines. Supplementary Fig. S3. Effect of treatment with IST5-002 in Ph like leukemia cells. Supplementary Fig. S4. Effect of IST5-002 on G-CSF-mobilized peripheral blood normal CD34+ hematopoietic cells. Supplementary Fig. S5. Effect of BIM silencing on apoptosis of STAT5-silenced Ph+ ALL cells. Supplementary Fig. S6. qPCR analysis of STAT5-regulated genes in untreated and Doxy-treated shScramble- or shSTAT5-BV173 cells. Supplementary Fig. S7. Levels of PIM1-s in STAT5-silenced Ph+ ALL lines and correlation with STAT5 expression in primary Ph+ ALL cells. Supplementary Fig. S8. Ectopic expression of PIM-1 does not rescue the apoptosis of STAT5-silenced BV173 cells. Supplementary Fig. S9. Effect of AZD1208 and Sabutoclax on cell growth and survival of Ph+ leukemia cell lines. Supplementary Fig. S10. Effects of the AZD1208- Sabutoclax combination on the growth of Ph+ leukemia cell lines. Supplementary Fig. S11. Effects of the AZD1208, Sabutoclax, or the drug combination on colony formation of Ph-like cell lines.

Primary human Ph+ ALL samples information.

PDF file - 146K, Effect of TAT-SNAG peptide on migration, invasion and proliferation of p53-null HCT116 cells

Figure S4. MIR300 anti-proliferative activity accounts for BMM-induced LSC entry into quiescence. A, Structure of 14q32 DLK1-DIO3 genomic imprinted locus hosting the MEG3-regulated human MIR300. B, MIR300 levels in 5-Aza- or DMSO-treated (24h) Ph+ cells. C, Effect of hypoxia on proliferation of CFSE+CD34+ CML-BC cells. D, left: Effect of MSC (HS-5)-derived CM on LAMA-84 proliferation expressed as fold changes of CFSE mean of fluorescence intensity (MFI)+/-SEM; middle: pro-apoptotic effect of PAD (FTY720; 2.5uM) and DMSO (control) on HS-5-cultured LAMA-84 cells; right: Effect of MSC (HS-5)-derived CM BCR-ABL1 expression (anti-ABL1) and activity anti-PY), phospho-BCR-ABL1, JAK2 expression and activity JAK2 Y1007/1008, PP2A activity (pPP2AY307 inactive form) and GRB2 used as a control (blots are representative of three independent experiments). E, Levels of C/EBPbeta and GRB2 mRNA and protein in HS-5 cells exposed to hypoxia (48h; 1% O2). F, Effect of neutralizing TGFbeta antibody (anti-TGFb Ab; 48h, 1.25 μg/ml) on MIR300 levels in CD34+ CML-BC cells. G, Effect of ectopic C/EBPalpha (MigR1-deltauORF-C/EBPalpha-HA) and C/EBPbeta (MigR1-C/EBPB-ERTAM) on MIR300 levels in K562 cells. Immunoblot shows levels of C/EBPbeta and GRB2 in normoxic and hypoxic K562 cells.

PDF file - 76K

Range values of controls for the indicated experiments

Figure S3. MIR300 acts as master PP2A activator and inhibitor of G1/S transition. A, (left) Additional effects of MIR300 on SET, CCND2 and CDK6 expression; (right) KEGG/GO analysis of MIR300 effects on signal transduction pathways. B, CSmiRTar (filters: bone marrow normal and myeloid leukemia cells) and miRDIP-ComiR integrated analyses show functional clustering of predicted/validated MIR300 targets.

Ectopic expression of CAAT/enhancer binding protein α (C/EBPα) in p210BCR/ABL-expressing cells induces granulocytic differentiation, inhibits proliferation, and suppresses leukemogenesis. To dissect the molecular mechanisms underlying these biological effects, C/EBPα-regulated genes were identified by microarray analysis in 32D-p210BCR/ABL cells. One of the genes whose expression was activated by C/EBPα in a DNA binding–dependent manner in BCR/ABL-expressing cells is the transcriptional repressor Gfi-1. We show here that C/EBPα interacts with a functional C/EBP binding site in the Gfi-1 5′-flanking region and enhances the promoter activity of Gfi-1. Moreover, in K562 cells, RNA interference–mediated downregulation of Gfi-1 expression partially rescued the proliferation-inhibitory but not the differentiation-inducing effect of C/EBPα. Ectopic expression of wild-type Gfi-1, but not of a transcriptional repressor mutant (Gfi-1P2A), inhibited proliferation and markedly suppressed colony formation but did not induce granulocytic differentiation of BCR/ABL-expressing cells. By contrast, Gfi-1 short hairpin RNA–tranduced CD34+ chronic myeloid leukemia cells were markedly more clonogenic than the scramble-transduced counterpart. Together, these studies indicate that Gfi-1 is a direct target of C/EBPα required for its proliferation and survival-inhibitory effects in BCR/ABL-expressing cells. Cancer Res; 70(20); 7949–59. ©2010 AACR.

Figure S2. KEGG/GO analysis of MIR300 on PP2A-regulated signal transduction pathways. Cartoon shows the SET-dependent PP2A Inhibitory pathway in CML and the pleiotropic inhibitory effect of PP2A activation on validated and predicted MIR300 targets regulating G1/S cell cycle transition, Wnt-beta-catenin, TGFbeta, JAK-STAT, PI-3K-Akt, RAS-MAPK and Notch signaling pathways.

Figure S5. BMM-induced MIR300 anti-proliferative and PP2A-activating functions impair NK cell immune-response. A, PP2A-dependent regulation of MIR300 and miR-155 validated pathways predicted to occur also in the bone marrow endosteal niche. B, Effect of hypoxia (1% O2, 7 days) on CFSE+NK cell proliferation (% CFSE mean of fluorescence). C, HS-5 exosomal miRNAs reported as negative regulators of NK cell proliferation/activity and their experimentally validated targets. miRNA RNAseq was performed on an Illumina platform using libraries derived from 100 ng RNA/sample from HS-5 exosome purifications (n=3). D, Precursor (pre-miR-155) miR-155 (BIC) levels in resting and IL-12/IL-18 (18h)-stimulated NK cells exposed (48h) to HS-5 exosomes. E, Effect of hypoxia (1% O2) and HS-5 CM (48h) on TUG1 expression in CD56+CD3- primary NK and NK-92 cells. F, Effect of CpG-TUG1-shRNA and CpG-scramble (200-500 nM, 5 days) on IL-2-induced NK cell proliferation (% cell number). Data are represented as mean+/-SEM.

Supplementary Figure 1 from Expression of the Transcriptional Repressor Gfi-1 Is Regulated by C/EBPα and Is Involved in Its Proliferation and Colony Formation–Inhibitory Effects in p210BCR/ABL-Expressing Cells

Figure S1. MIR300 activity in quiescent leukemic stem and progenitor cells. CFC-replating assays shows effects of lentiviral-mediated ectopic MIR300 expression, 250 nM and 500 nM CpG-miR-300 on serial replating activity (2nd replating) of leukemic chronic and acute CML and normal UCB CD34+CD38- HSC-enriched cell fractions. Infection with lentiviral empty vector and treatment with CpG-anti-MIR300 and CpG-scramble served as controls.

PDF file - 153K, Effect of LSD1 silencing on migration and invasion of HCT116 cells

PDF file - 5MB, Effect of Slug silencing on invasion and EMT markers expression of HTLA230 cells

Despite treatment with intensive chemotherapy, acute myelogenous leukemia (AML) remains an aggressive malignancy with a dismal outcome in most patients. We found that AML cells exhibit an unusually rapid accumulation of the repressive histone mark H3K27me3 on nascent DNA. In cell lines, primary cells and xenograft mouse models, inhibition of the H3K27 histone methyltransferase EZH2 to decondense the H3K27me3-marked chromatin of AML cells enhanced chromatin accessibility and chemotherapy-induced DNA damage, apoptosis, and leukemia suppression. These effects were further promoted when chromatin decondensation of AML cells was induced upon S-phase entry after release from a transient G1 arrest mediated by CDK4/6 inhibition. In the p53-null KG-1 and THP-1 AML cell lines, EZH2 inhibitor and doxorubicin cotreatment induced transcriptional reprogramming that was, in part, dependent on derepression of H3K27me3-marked gene promoters and led to increased expression of cell death–promoting and growth-inhibitory genes. In conclusion, decondensing H3K27me3-marked chromatin by EZH2 inhibition represents a promising approach to improve the efficacy of DNA-damaging cytotoxic agents in patients with AML. This strategy might allow for a lowering of chemotherapy doses, with a consequent reduction of treatment-related side effects in elderly patients with AML or those with significant comorbidities. Significance: Pharmacological inhibition of EZH2 renders DNA of AML cells more accessible to cytotoxic agents, facilitating leukemia suppression with reduced doses of chemotherapy. See related commentary by Adema and Colla, p. 359

Ph+ ALL is a poor-prognosis leukemia subtype driven by the BCR-ABL1 oncogene, either the p190- or the p210-BCR/ABL isoform in a 70:30 ratio. Tyrosine Kinase inhibitors (TKIs) are the drugs of choice in the therapy of Ph+ ALL. In combination with standard chemotherapy, TKIs have markedly improved the outcome of Ph+ ALL, in particular if this treatment is followed by bone marrow transplantation. However, resistance to TKIs develops with high frequency, causing leukemia relapse that results in

Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that

Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that MIR300 has antiproliferative and PP2A-activating functions that are dose dependently differentially induced by CCND2/CDK6 and SET inhibition, respectively. MIR300 is upregulated in CML LSCs and NK cells by bone marrow microenvironment (BMM) signals to induce quiescence and impair immune response, respectively. Conversely, BCR-ABL1 downregulates MIR300 in CML progenitors to prevent growth arrest and PP2A-mediated apoptosis. Quiescent LSCs escape apoptosis by upregulating TUG1 long noncoding RNA that uncouples and limits MIR300 function to cytostasis. Genetic and pharmacologic MIR300 modulation and/or PP2A-activating drug treatment restore NK cell activity, inhibit BMM-induced growth arrest, and selectively trigger LSC apoptosis in vitro and in patient-derived xenografts; hence, the importance of MIR300 and PP2A activity for CML development and therapy. Significance: Tumor-naïve microenvironment–induced MIR300 is the only tumor suppressor miRNA that induces CML LSC quiescence while inhibiting NK cell antitumor immune response, and CML LSC/progenitor cell apoptosis through its anti-proliferative and PP2A-activating functions, respectively. Thus, the importance of MIR300 and PP2A-activating drugs for formation/survival and eradication of drug-resistant CML LSCs, respectively. See related commentary by Broxmeyer, p. 13. This article is highlighted in the In This Issue feature, p. 5

Combining standard cytotoxic chemotherapy with BCR-ABL1 tyrosine kinase inhibitors (TKI) has greatly improved the upfront treatment of patients with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). However, due to the development of drug resistance through both BCR-ABL1–dependent and -independent mechanisms, prognosis remains poor. The STAT5 transcription factor is activated by BCR-ABL1 and by JAK2-dependent cytokine signaling; therefore, inhibiting its activity could address both mechanisms of resistance in Ph+ ALL. We show here that genetic and pharmacologic inhibition of STAT5 activity suppresses cell growth, induces apoptosis, and inhibits leukemogenesis of Ph+ cell lines and patient-derived newly diagnosed and relapsed/TKI-resistant Ph+ ALL cells ex vivo and in mouse models. STAT5 silencing decreased expression of the growth-promoting PIM-1 kinase, the apoptosis inhibitors MCL1 and BCL2, and increased expression of proapoptotic BIM protein. The resulting apoptosis of STAT5-silenced Ph+ BV173 cells was rescued by silencing of BIM or restoration of BCL2 expression. Treatment of Ph+ ALL cells, including samples from relapsed/refractory patients, with the PIM kinase inhibitor AZD1208 and/or the BCL2 family antagonist Sabutoclax markedly suppressed cell growth and leukemogenesis ex vivo and in mice. Together, these studies indicate that targeting STAT5 or STAT5-regulated pathways may provide a new approach for therapy development in Ph+ ALL, especially the relapsed/TKI-resistant disease. Significance:Suppression of STAT5 by BCL2 and PIM kinase inhibitors reduces leukemia burden in mice and constitutes a new potential therapeutic approach against Ph+ ALL, especially in tyrosine kinase inhibitor-resistant disease. Cancer Res; 78(20); 5793–807. ©2018 AACR.

Expression of the cell cycle regulatory gene CDK6 is required for Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) cell growth, whereas expression of the closely related CDK4 protein is dispensable. Moreover, CDK6 silencing is more effective than treatment with the dual CDK4/6 inhibitor palbociclib in suppressing Ph+ ALL in mice, suggesting that the growth-promoting effects of CDK6 are, in part, kinase-independent in Ph+ ALL. Accordingly, we developed CDK4/6–targeted proteolysis-targeting chimeras (PROTACs) that inhibit CDK6 enzymatic activity in vitro, promote the rapid and preferential degradation of CDK6 over CDK4 in Ph+ ALL cells, and markedly suppress S-phase cells concomitant with inhibition of CDK6-regulated phospho-RB and FOXM1 expression. No such effects were observed in CD34+ normal hematopoietic progenitors, although CDK6 was efficiently degraded. Treatment with the CDK6-degrading PROTAC YX-2-107 markedly suppressed leukemia burden in mice injected with de novo or tyrosine kinase inhibitor–resistant primary Ph+ ALL cells, and this effect was comparable or superior to that of the CDK4/6 enzymatic inhibitor palbociclib. These studies provide “proof of principle” that targeting CDK6 with PROTACs that inhibit its enzymatic activity and promote its degradation represents an effective strategy to exploit the “CDK6 dependence” of Ph+ ALL and, perhaps, of other hematologic malignancies. Moreover, they suggest that treatment of Ph+ ALL with CDK6-selective PROTACs would spare a high proportion of normal hematopoietic progenitors, preventing the neutropenia induced by treatment with dual CDK4/6 inhibitors.

Drug-resistance of tumor-initiating cells, impaired NK cell immune-response, PP2A loss-of-function and aberrant miRNA expression are cancer features resulting from microenvironmental- and tumor-specific signals. Here we report that genomic-imprintedMIR300is a cell context-independent dual function tumor suppressor which is upregulated in quiescent leukemic stem (LSC) and NK cells by microenvironmental signals to induce quiescence and impair immune-response, respectively, but inhibited in CML and AML proliferating blasts to prevent PP2A-induced apoptosis.MIR300anti-proliferative and PP2A-activating functions are differentially activated through dose-dependent CCND2/CDK6 and SET inhibition, respectively. LSCs escape PP2A-mediated apoptosis through TUG1 lncRNA that uncouples and limitsMIR300functions to cytostasis by regulating unbound-MIR300levels. HaltingMIR300homeostasis restores NK cell activity and suppresses leukemic but not normal hematopoiesis by eradicating nearly all LSCs. Thus,MIR300tumor suppressor activity is essential and therapeutically important for LSC-driven leukemias.

MicroRNAs, non-coding regulators of gene expression, are likely to function as important downstream effectors of many transcription factors including MYB. Optimal levels of MYB are required for transformation/maintenance of BCR-ABL-expressing cells. We investigated whether MYB silencing modulates microRNA expression in Philadelphia-positive (Ph+) leukemia cells and if MYB-regulated microRNAs are important for the "MYB addiction" of these cells. Thirty-five microRNAs were modulated by MYB silencing in lymphoid and erythro-myeloid chronic myeloid leukemia-blast crisis BV173 and K562 cells; 15 of these were concordantly modulated in both lines. We focused on the miR-17-92 cluster because of its oncogenic role in tumors and found that: i) it is a direct MYB target; ii) it partially rescued the impaired proliferation and enhanced apoptosis of MYB-silenced BV173 cells. Moreover, we identified FRZB, a Wnt/beta-catenin pathway inhibitor, as a novel target of the miR-17-92 cluster. High expression of MYB in blast cells from 2 Ph+ leukemia patients correlated positively with the miR-17-92 cluster and inversely with FRZB. This expression pattern was also observed in a microarray dataset of 122 Ph+ acute lymphoblastic leukemias. In vivo experiments in NOD scid gamma mice injected with BV173 cells confirmed that FRZB functions as a Wnt/beta-catenin inhibitor even as they failed to demonstrate that this pathway is important for BV173-dependent leukemogenesis. These studies illustrate the global effects of MYB expression on the microRNAs profile of Ph+ cells and supports the concept that the "MYB addiction" of these cells is, in part, caused by modulation of microRNA-regulated pathways affecting cell proliferation and survival.

BackgroundThe Philadelphia (Ph) chromosome generates aberrant BCR-ABL constitutively active proteins, and represents the most frequent cytogenetic abnormality in acute lymphoblastic leukemia (ALL). BCR-ABL is an ideal target for Ph+ leukemia treatment, thus tyrosine kinase inhibitors (TKIs) were developed as therapeutic drugs. Although such drugs have markedly improved the Ph+ ALL outcome, many patients develop resistance to TKIs treatment, frequently associated with point mutations or amplification of the BCR-ABL gene, but also with the activation of BCR-ABL-independent pathways. MicroRNAs (miRNAs) play a critical role in pharmacogenomics regulating the expression of genes essential for drug activity. Aberrant miRNAs expression may be involved in BCR-ABL-dependent/independent TKI-resistance mechanisms, among them members of miR-17-92 cluster. We previously showed that modulation of the oncogenic miR-17-92 cluster, a direct target of the transcription factor MYB, rescued the impaired proliferation and enhanced apoptosis of MYB-silenced Ph+ ALL cells, indicating that de-regulated miRNAs are involved in the MYB “addiction” of Ph+ ALL. Members of miR-17-92 cluster were reported to be involved in TKI-resistance, but MYB expression is not affected by TKIs treatment in Ph+ cells, suggesting that MYB-dependent pathways could have a role in BCR-ABL-independent mechanisms of drug resistance. AimTo evaluate the relationship between the miR-17-92 cluster, its target genes and resistance to tyrosine kinase inhibitors. ResultsWe assessed the cell survival in miR-17-92-overexpressing compared to empty vector (EV) Ph+ ALL cells, after TKIs treatment. The results showed that miR-17-92 cluster contributes in promoting the survival of Ph+ ALL TKI-treated cells. We evaluated the cell death through the apoptotic markers, observing that overexpressing miR-17-92 cells showed less apoptosis than EV cells, after TKI-treatment. We analyzed the MYB protein and miR-17-92 expression in miR-overexpressed compared to EV cells. We found that both MYB and miR-17-92 levels are not modulated by TKI treatment indicating a possible activation of alternative pathways under their control operating despite effective TKI inhibition of BCR-ABL1. Thus, our data underscore the importance of investigating the role of miR-17-92 cluster in TKI-resistant Ph+ leukemia cells. This might help to reveal a strategy for new therapeutic approaches in drug resistance. ConclusionsThese data suggest that the miR-17-92 cluster might exert anti-apoptotic and pro-proliferative effects in Ph+ acute lymphoblastic leukemia cells after TKIs treatment and investigating its biological role may lead to future breakthroughs in TKI-resistant Ph+ leukemia. Citation Format: Manuela Spagnuolo, Giulia Regazzo, Andrea Sacconi, Ana B. Díaz Méndez, Bruno Calabretta, Maria G. Rizzo. Investigating the role of the oncogenic miR-17-92 cluster as involved in TKI-resistance enhancing the survival of MYB-dependent Ph+ acute lymphoblastic leukemia cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2538.

Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ ALL) is currently treated with BCR-ABL1 tyrosine kinase inhibitors (TKI) in combination with chemotherapy. However, most patients develop resistance to TKI through BCR-ABL1–dependent and –independent mechanisms. Newly developed TKI can target Ph+ ALL cells with BCR-ABL1–dependent resistance; however, overcoming BCR-ABL1–independent mechanisms of resistance remains challenging because transcription factors, which are difficult to inhibit, are often involved. We show here that (i) the growth of Ph+ ALL cell lines and primary cells is highly dependent on MYB-mediated transcriptional upregulation of CDK6, cyclin D3, and BCL2, and (ii) restoring their expression in MYB-silenced Ph+ ALL cells rescues their impaired proliferation and survival. Levels of MYB and CDK6 were highly correlated in adult Ph+ ALL (P = 0.00008). Moreover, Ph+ ALL cells exhibited a specific requirement for CDK6 but not CDK4 expression, most likely because, in these cells, CDK6 was predominantly localized in the nucleus, whereas CDK4 was almost exclusively cytoplasmic. Consistent with their essential role in Ph+ ALL, pharmacologic inhibition of CDK6 and BCL2 markedly suppressed proliferation, colony formation, and survival of Ph+ ALL cells ex vivo and in mice. In summary, these findings provide a proof-of-principle, rational strategy to target the MYB "addiction" of Ph+ ALL. Significance: MYB blockade can suppress Philadelphia chromosome-positive leukemia in mice, suggesting that this therapeutic strategy may be useful in patients who develop resistance to imatinib and other TKIs used to treat this disease. Cancer Res; 78(4); 1097–109. ©2017 AACR.

The transcription factor C/EBPα is required for granulocytic differentiation of normal myeloid progenitors and is frequently inactivated in acute myeloid leukemia (AML) cells. Ectopic expression of C/EBPα in AML cells suppresses proliferation and induces differentiation suggesting that restoring C/EBPα expression/activity in AML cells could be therapeutically useful. Unfortunately, current approaches of gene or protein delivery in leukemic cells are unsatisfactory. However, "drug repurposing" is becoming a very attractive strategy to identify potential new uses for existing drugs. In this study, we assessed the biological effects of candidate C/EBPα-mimetics identified by interrogation of the Connectivity Map database. We found that amantadine, an antiviral and anti-Parkinson agent, induced a monocyte-macrophage-like differentiation of HL60, U937, Kasumi-1 myeloid leukemia cell lines, as indicated by morphology and differentiation antigen expression, when used in combination with suboptimal concentration of all trans retinoic acid (ATRA) or Vit D3. The effect of amantadine depends, in part, on increased activity of the vitamin D receptor (VDR), since it induced VDR expression and amantadine-dependent monocyte-macrophage differentiation of HL60 cells was blocked by expression of dominant-negative VDR. These results reveal a new function for amantadine and support the concept that screening of the Connectivity Map database can identify small molecules that mimic the effect of transcription factors required for myelo-monocytic differentiation.

Most triple-negative breast cancers (TNBCs) exhibit gene expression patterns associated with epithelial-to-mesenchymal transition (EMT), a feature that correlates with a propensity for metastatic spread. Overexpression of the EMT regulator Slug is detected in basal and mesenchymal-type TNBCs and is associated with reduced E-cadherin expression and aggressive disease. The effects of Slug depend, in part, on the interaction of its N-terminal SNAG repressor domain with the chromatin-modifying protein lysine demethylase 1 (LSD1); thus, we investigated whether tranylcypromine [also known as trans-2-phenylcyclopropylamine hydrochloride (PCPA) or Parnate], an inhibitor of LSD1 that blocks its interaction with Slug, suppresses the migration, invasion, and metastatic spread of TNBC cell lines. We show here that PCPA treatment induces the expression of E-cadherin and other epithelial markers and markedly suppresses migration and invasion of TNBC cell lines MDA-MB-231 and BT-549. These effects were phenocopied by Slug or LSD1 silencing. In two models of orthotopic breast cancer, PCPA treatment reduced local tumor growth and the number of lung metastases. In mice injected directly in the blood circulation with MDA-MB-231 cells, PCPA treatment or Slug silencing markedly inhibited bone metastases but had no effect on lung infiltration. Thus, blocking Slug activity may suppress the metastatic spread of TNBC and, perhaps, specifically inhibit homing/colonization to the bone.

Mesalazine (5-ASA) is an aminosalicylate anti-inflammatory drug capable of inducing μ-protocadherin, a protein expressed by colorectal epithelial cells that is downregulated upon malignant transformation. Treatment with 5-ASA restores μ-protocadherin expression and promotes the sequestration of β-catenin to the plasma membrane. Here, we show that 5-ASA–induced μ-protocadherin expression is directly regulated by the KLF4 transcription factor. In addition, we suggest the existence of a dual mechanism whereby 5-ASA–mediated β-catenin inhibition is caused by μ-protocadherin–dependent sequestration of β-catenin to the plasma membrane and by the direct binding of KLF4 to β-catenin. In addition, we found that 5-ASA treatment suppresses the expression of miR-130a and miR-135b, which target KLF4 mRNA, raising the possibility that this mechanism is involved in the increased expression of KLF4 induced by 5-ASA. Cancer Prev Res; 11(8); 503–10. ©2018 AACR.