Selective inhibition of Ph-positive ALL cell growth through kinase-dependent and -independent effects by CDK6-specific PROTACs.

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.

Transcriptional activation of the miR-17-92 cluster is involved in the growth-promoting effects of MYB in human Ph-positive leukemia cells.

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 erythromyeloid 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/ß-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/ß-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.

A phase I study of the combination of palbociclib and dexamethasone for the treatment of relapsed or refractory B-cell acute lymphoblastic leukemia.

PURPOSE: Despite advances in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), outcomes for relapsed/refractory (R/R) disease remain poor. Preclinical studies suggest that the combination of the CDK4/6 inhibitor palbociclib and dexamethasone may be effective in targeting leukemic cell growth. We conducted a phase I study of escalating doses of palbociclib in combination with dexamethasone in adults with R/R B-ALL. METHODS: Cycle 1 consisted of single agent palbociclib given for 7 days and continued for 28 additional days in combination with dexamethasone 20 mg daily. Palbociclib dosing began at 100 mg daily. Patients with a response were eligible for maintenance consisting of 1 week of palbociclib plus dexamethasone (20 mg daily × 2 days, 16 mg daily × 2 days, 12 mg daily × 2 days, 6 mg daily × 1 day), followed by 3 weeks of palbociclib alone. Safety, efficacy, and the expression of phospho-RB and c-MYB/BCL-2 were measured. CONCLUSIONS: Seven patients were treated on study before it was closed early due to slow accrual. No dose limiting toxicities were identified. One patient had a complete response with incomplete hematologic recovery, suggesting possible efficacy of the treatment. Reduction in CD34+ cells, p-RB, c-MYB, and BCL-2 expression also suggested on-target therapy effects.

Dnmt3a-Mutant Hematopoietic Stem Cell Rewire IFNγ Signaling to Gain Clonal Advantage.

SUMMARY: Dnmt3a-mutant stem cells gain a competitive advantage via upregulation of a Txnip-p53-p21 axis and protection from IFNγ induced exhaustion. See related article by Zhang et al., p. 220 (5).

Targeting Chemotherapy to Decondensed H3K27me3-Marked Chromatin of AML Cells Enhances Leukemia Suppression.

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.

Shedding Light on Mutant Clonal Dynamics and Cancer Risk in the Skin.

In this issue of Cancer Discovery, Fowler and colleagues conduct a thorough characterization of the dynamics of mutant clones in phenotypically normal human skin. Their results extend previous studies by showing that human skin is composed in large part of clones harboring mutations frequently observed in human cancer, while at the same time they uncover a previously unappreciated biological heterogeneity among nearby clones and across different body sites.See related article by Fowler et al., p. 340.

Targeting the CDK6 Dependence of Ph+ Acute Lymphoblastic Leukemia.

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 <5-year overall survival. Thus, new therapies are needed to address relapsed/TKI-resistant Ph+ ALL. We have shown that expression of cell cycle regulatory kinase CDK6, but not of the highly related CDK4 kinase, is required for the proliferation and survival of Ph+ ALL cells. Comparison of leukemia suppression induced by treatment with the clinically-approved dual CDK4/6 inhibitor palbociclib versus CDK6 silencing revealed that the latter treatment was markedly more effective, probably reflecting inhibition of CDK6 kinase-independent effects. Thus, we developed CDK4/6-targeted proteolysis-targeting chimeras (PROTACs) that preferentially degrade CDK6 over CDK4. One compound termed PROTAC YX-2-107, which degrades CDK6 by recruiting the Cereblon ubiquitin ligase, markedly suppressed leukemia burden in mice injected with de novo or TKI-resistant Ph+ ALL. The effect of PROTAC YX-2-107 was comparable or superior to that of palbociclib. The development of CDK6-selective PROTACs represents an effective strategy to exploit the "CDK6 dependence" of Ph+ ALL cells while sparing a high proportion of normal hematopoietic progenitors that depend on both CDK6 and CDK6 for their survival. In combination with other agents, CDK6-selective PROTACs may be valuable components of chemotherapy-free protocols for the therapy of Ph+ ALL and other CDK6-dependent hematological malignancies.

Chronic interleukin-1 exposure triggers selection for Cebpa-knockout multipotent hematopoietic progenitors.

The early events that drive myeloid oncogenesis are not well understood. Most studies focus on the cell-intrinsic genetic changes and how they impact cell fate decisions. We consider how chronic exposure to the proinflammatory cytokine, interleukin-1ß (IL-1ß), impacts Cebpa-knockout hematopoietic stem and progenitor cells (HSPCs) in competitive settings. Surprisingly, we found that Cebpa loss did not confer a hematopoietic cell-intrinsic competitive advantage; rather chronic IL-1ß exposure engendered potent selection for Cebpa loss. Chronic IL-1ß augments myeloid lineage output by activating differentiation and repressing stem cell gene expression programs in a Cebpa-dependent manner. As a result, Cebpa-knockout HSPCs are resistant to the prodifferentiative effects of chronic IL-1ß, and competitively expand. We further show that ectopic CEBPA expression reduces the fitness of established human acute myeloid leukemias, coinciding with increased differentiation. These findings have important implications for the earliest events that drive hematologic disorders, suggesting that chronic inflammation could be an important driver of leukemogenesis and a potential target for intervention.

Targeting STAT5 or STAT5-Regulated Pathways Suppresses Leukemogenesis of Ph+ Acute Lymphoblastic Leukemia.

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.

Targeting CDK6 and BCL2 Exploits the "MYB Addiction" of Ph+ Acute Lymphoblastic Leukemia.

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.

Semaphorin 5A drives melanoma progression: role of Bcl-2, miR-204 and c-Myb.

BACKGROUND: Melanoma, the most aggressive form of skin cancer, is characterized by high rates of metastasis, drug resistance and mortality. Here we investigated the role of Semaphorin 5A (Sema5A) on the properties associated with melanoma progression and the factors involved in Sema5A regulation. METHODS: Western blotting, qRT-PCR, Chromatin immunoprecipitation (ChIP) assay, immunohistochemistry of melanoma patient specimens and xenograft tissues, in vitro Transwell assay for cell migration and invasion evaluation, in vitro capillary-like structure formation analysis. RESULTS: A significant correlation of Sema5A mRNA expression and melanoma progression was observed by analyzing GEO profile dataset. Endogenous Sema5A protein was detected in 95% of human melanoma cell lines tested, in 70% of metastatic specimens from patients affected by melanoma, and 16% of in situ melanoma specimens showed a focal positivity. We demonstrated that Sema5A regulates in vitro cell migration and invasion and the formation of vasculogenic structures. We also found an increase of Sema5A at both mRNA and protein level after forced expression of Bcl-2. By use of transcriptional and proteasome inhibitors, we showed that Bcl-2 increases the stability of Sema5A mRNA and protein. Moreover, by ChIP we demonstrated that Sema5A expression is under the control of the transcription factor c-Myb and that c-Myb recruitment on Sema5A promoter is increased after Bcl-2 overexpression. Finally, a concomitant decrease in the expression of Sema5A, Bcl-2 and c-Myb proteins was observed in melanoma cells after miR-204 overexpression. CONCLUSION: Overall our data provide evidences supporting the role of Sema5A in melanoma progression and the involvement of Bcl-2, miR-204 and c-Myb in regulating its expression.

Structure of Nascent Chromatin Is Essential for Hematopoietic Lineage Specification.

The role of chromatin structure in lineage commitment of multipotent hematopoietic progenitors (HPCs) is presently unclear. We show here that CD34+ HPCs possess a post-replicative chromatin globally devoid of the repressive histone mark H3K27me3. This H3K27-unmodified chromatin is required for recruitment of lineage-determining transcription factors (TFs) C/EBPα, PU.1, and GATA-1 to DNA just after DNA replication upon cytokine-induced myeloid or erythroid commitment. Blocking DNA replication or increasing H3K27me3 levels prevents recruitment of these TFs to DNA and suppresses cytokine-induced erythroid or myeloid differentiation. However, H3K27me3 is rapidly associated with nascent DNA in more primitive human and murine HPCs. Treatment of these cells with instructive cytokines leads to a significant delay in accumulation of H3K27me3 in nascent chromatin due to activity of the H3K27me3 demethylase UTX. Thus, HPCs utilize special mechanisms of chromatin modification for recruitment of specific TFs to DNA during early stages of lineage specification.

Celecoxib inhibits proliferation and survival of chronic myelogeous leukemia (CML) cells via AMPK-dependent regulation of ß-catenin and mTORC1/2.

CML is effectively treated with tyrosine kinase inhibitors (TKIs). However, the efficacy of these drugs is confined to the chronic phase of the disease and development of resistance to TKIs remains a pressing issue. The anti-inflammatory COX2 inhibitor celecoxib has been utilized as anti-tumour drug due to its anti-proliferative activity. However, its effects in hematological malignancies, in particular CML, have not been investigated yet. Thus, we tested biological effects and mechanisms of action of celecoxib in Philadelphia-positive (Ph+) CML and ALL cells.We show here that celecoxib suppresses the growth of Ph+ cell lines by increasing G1-phase and apoptotic cells and reducing S- and G2-phase cells. These effects were independent of COX2 inhibition but required the rapid activation of AMP-activated protein kinase (AMPK) and the consequent inhibition mTORC1 and 2. Treatment with celecoxib also restored GSK3ß function and led to down-regulation of ß-catenin activity through transcriptional and post-translational mechanisms, two effects likely to contribute to Ph+ cell growth suppression by celecoxib.Celecoxib inhibited colony formation of TKI-resistant Ph+ cell lines including those with the T315I BCR-ABL mutation and acted synergistically with imatinib in suppressing colony formation of TKI-sensitive Ph+ cell lines. Finally, it suppressed colony formation of CD34+ cells from CML patients, while sparing most CD34+ progenitors from healthy donors, and induced apoptosis of primary Ph+ ALL cells.Together, these findings indicate that celecoxib may serve as a COX2-independent lead compound to simultaneously target the mTOR and ß-catenin pathways, key players in the resistance of CML stem cells to TKIs.

Structure-Based Screen Identifies a Potent Small Molecule Inhibitor of Stat5a/b with Therapeutic Potential for Prostate Cancer and Chronic Myeloid Leukemia.

Bypassing tyrosine kinases responsible for Stat5a/b phosphorylation would be advantageous for therapy development for Stat5a/b-regulated cancers. Here, we sought to identify small molecule inhibitors of Stat5a/b for lead optimization and therapy development for prostate cancer and Bcr-Abl-driven leukemias. In silico screening of chemical structure databases combined with medicinal chemistry was used for identification of a panel of small molecule inhibitors to block SH2 domain-mediated docking of Stat5a/b to the receptor-kinase complex and subsequent phosphorylation and dimerization. We tested the efficacy of the lead compound IST5-002 in experimental models and patient samples of two known Stat5a/b-driven cancers, prostate cancer and chronic myeloid leukemia (CML). The lead compound inhibitor of Stat5-002 (IST5-002) prevented both Jak2 and Bcr-Abl-mediated phosphorylation and dimerization of Stat5a/b, and selectively inhibited transcriptional activity of Stat5a (IC50 = 1.5µmol/L) and Stat5b (IC50 = 3.5 µmol/L). IST5-002 suppressed nuclear translocation of Stat5a/b, binding to DNA and Stat5a/b target gene expression. IST5-002 induced extensive apoptosis of prostate cancer cells, impaired growth of prostate cancer xenograft tumors, and induced cell death in patient-derived prostate cancers when tested ex vivo in explant organ cultures. Importantly, IST5-002 induced robust apoptotic death not only of imatinib-sensitive but also of imatinib-resistant CML cell lines and primary CML cells from patients. IST5-002 provides a lead structure for further chemical modifications for clinical development for Stat5a/b-driven solid tumors and hematologic malignancies.