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

Philadelphia chromosome–positive acute lymphoblastic leukemia (Phþ 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 Phþ 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 Phþ 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 Phþ ALL cells rescues their impaired proliferation and survival. Levels of MYB and CDK6 were highly correlated in adult Phþ ALL (P ¼ 0.00008). Moreover, Phþ 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 Phþ ALL, pharmacologic inhibition of CDK6 and BCL2 markedly suppressed proliferation, colony formation, and survival of Phþ ALL cells ex vivo and in mice. In summary, these findings provide a proof-of-principle, rational strategy to target the MYB addiction of Phþ ALL. © 2017 American Association for Cancer Research

Monocyte-macrophage differentiation of acute myeloid leukemia cell lines by small molecules identified through interrogation of the Connectivity Map database

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

Suppression of Invasion and Metastasis of Triple-Negative Breast Cancer Lines by Pharmacological or Genetic Inhibition of Slug Activity

AbstractMost 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

The p53 Codon 72 Pro/Pro Genotype Identifies Poor-Prognosis Neuroblastoma Patients: Correlation with Reduced Apoptosis and Enhanced Senescence by the p53-72P Isoform.

The p53 gene is rarely mutated in neuroblastoma, but codon 72 polymorphism that modulates its proapoptotic activity might influence cancer risk and clinical outcome. We investigated whether this polymorphism affects neuroblastoma risk and disease outcome and assessed the biologic effects of the p53-72R and p53-72P isoforms in p53-null cells. Comparison of 288 healthy subjects and 286 neuroblastoma patients revealed that the p53-72 polymorphism had no significant impact on the risk of developing neuroblastoma; however, patients with the Pro/Pro genotype had a shorter survival than those with the Arg/Arg or the Arg/Pro genotypes even in the stage 3 and 4 subgroup without MYCN amplification. By Cox regression analysis, the p53 Pro/Pro genotype seems to be an independent marker of poor prognosis (hazard ratio = 2.74; 95% confidence interval = 1.14\u20136.55, P = .014) together with clinical stage, MYCN status, and age at diagnosis. In vitro, p53-72P was less effective than p53-72R in inducing apoptosis and inhibiting survival of p53-null LAN-1 cells treated with etoposide, topotecan, or ionizing radiation but not taxol. By contrast, p53-72P was more effective in promoting p21-dependent accelerated senescence, alone or in the presence of etoposide. Thus, the p53-72 Pro/Pro genotype might be a marker of poor outcome independent of MYCN amplification, possibly improving risk stratification. Moreover, the lower apoptosis and the enhanced accelerated senescence by the p53-72P isoform in response to DNA damage suggest that patients with neuroblastoma with the p53-72 Pro/Pro genotype may benefit from therapeutic protocols that do not rely only on cytotoxic drugs that function, in part, through p53 activation

Expression of p89c-Mybex9b, an alternatively spliced form of c-Myb, is required for proliferation and survival of p210BCR/ABL-expressing cells

The c-Myb gene encodes the p75c-Myb isoform and less-abundant proteins generated by alternatively spliced transcripts. Among these, the best known is pc-Mybex9b, which contains 121 additional amino acids between exon 9 and 10, in a domain involved in protein–protein interactions and negative regulation. In hematopoietic cells, expression of pc-Mybex9b accounts for 10–15% of total c-Myb; these levels may be biologically relevant because modest changes in c-Myb expression affects proliferation and survival of leukemic cells and lineage choice and frequency of normal hematopoietic progenitors. In this study, we assessed biochemical activities of pc-Mybex9b and the consequences of perturbing its expression in K562 and primary chronic myeloid leukemia (CML) progenitor cells. Compared with p75c-Myb, pc-Mybex9b is more stable and more effective in transactivating Myb-regulated promoters. Ectopic expression of pc-Mybex9b enhanced proliferation and colony formation and reduced imatinib (IM) sensitivity of K562 cells; conversely, specific downregulation of pc-Mybex9b reduced proliferation and colony formation, enhanced IM sensitivity of K562 cells and markedly suppressed colony formation of CML CD34+ cells, without affecting the levels of p75c-Myb. Together, these studies indicate that expression of the low-abundance pc-Mybex9b isoform has an important role for the overall biological effects of c-Myb in BCR/ABL-transformed cells

Targeting acute myeloid leukemia by drug-induced c-MYB degradation

Despite advances in our understanding of the molecular basis for particular subtypes of acute myeloid leukemia (AML), effective therapy remains a challenge for many individuals suffering from this disease. A significant proportion of both pediatric and adult AML patients cannot be cured and since the upper limits of chemotherapy intensification have been reached, there is an urgent need for novel therapeutic approaches. The transcription factor c-MYB has been shown to play a central role in the development and progression of AML driven by several different oncogenes, including mixed lineage leukemia (MLL)-fusion genes. Here, we have used a c-MYB gene expression signature from MLL-rearranged AML to probe the Connectivity Map database and identified mebendazole as a c-MYB targeting drug. Mebendazole induces c-MYB degradation via the proteasome by interfering with the heat shock protein 70 (HSP70) chaperone system. Transient exposure to mebendazole is sufficient to inhibit colony formation by AML cells, but not normal cord blood-derived cells. Furthermore, mebendazole is effective at impairing AML progression in vivo in mouse xenotransplantation experiments. In the context of widespread human use of mebendazole, our data indicate that mebendazole-induced c-MYB degradation represents a safe and novel therapeutic approach for AML

Rottura splenica “occulta” in paziente con mononucleosi infettiva

La rottura di milza può essere secondaria a traumi addominali (di solito chiusi) ovvero verificarsi spontaneamente su un organo sano o sede di alterazioni morfologiche secondarie a vari processi patologici. Tra le varie patologie responsabili di rottura occulta, le malattie infettive sono di gran lunga le più frequenti e tra queste un posto preminente ha la mononucleosi infettiva, che si complica con rottura splenica nello 0.5% dei casi, con una mortalità del 30%. Caso clinico. P.M., donna di 16 anni, è ricoverata in urgenza per addome acuto con anemizzazione ingravescente e incipiente instabilità emodinamica, cui si associa un quadro clinico suggestivo di mononucleosi infettiva, successivamente confermata sierologicamente e istologicamente. Confortati dal riscontro strumentale di grossa falda iperdensa pericapsulare splenica in continuità con spandimento peritoneale gravitario pelvico, abbiamo optato per una laparotomia esplorativa. Discussione. La rottura splenica nella mononucleosi si presenta invariabilmente con dolore in ipocondrio sinistro, raramente presente nelle forme non complicate; la sua insorgenza, pertanto, sia in paziente con recente diagnosi certa di mononucleosi sia in paziente con esclusivi reperti clinico-laboratoristici suggestivi di infezione acuta da virus di Epstein-Barr (EBV), obbliga all’esame ultrasonografico e/o alla TC di approfondimento, soprattutto nei casi in cui il dolore si associa a irradiazione alla spalla omolaterale (segno di Kehr), a segni di irritazione peritoneale e a instabilità emodinamica. Il trattamento della complicanza in esame è generalmente rappresentato dalla splenectomia in urgenza

I tumori stromali gastrointestinali (GIST). Esperienza personale su tre casi a localizzazione nel tenue mesenteriale trattati in urgenza

ummary: Gastrointestinal stromal tumors (GISTs). Personal experience on three cases of the tumors of the small intestine complicated and emergency surgically treated. M. Licursi, A. Bonsignore, F. Fiumara, M. Soliera, G. Grillone, G. Faro, G Pirrone, L.G. Angiò Introduction. GISTs, a new nosological entity recently described, represent a peculiar model of solid tumor: the identification of the molecular mechanism responsible for the oncogenesis led to the development of a new drug (imatinib) active on the specific molecular target, represented by the product of the mutated proto-oncogene c-kit which is a tirosyne kinasi receptor that becomes constitutively active by mutation. Surgical resection, nevertheless, is still the primary treatment and it has to be as complete as possible. These two treatments can be integrated. GISTs are not uniformly kit-positive, and they can be alternatively due to mutations of the PDGFRA gene or, in patients with neurofibromatosis type 1 (NF-1), to generally isolated mutations of the NF-1 gene. Patients and methods. We describe 3 cases of kit-positive GISTs of the small intestine (SISTs), complicated and emergency surgically treated: case 1 - 53 years, female, with small bowel obstruction and concomitant acute intestinal bleeding; case 2 - 71 years, male, with NF-1 and acute intestinal bleeding; case 3 - 47 years, male, with perforation of the Treitz tract. The first two cases have been treated with intestinal resection and immediate mechanical anastomosis; the third one with resection of the pedunculated tumor at its base, where is situated the perforation too. Conclusions. SISTs (20-30%), with little or no symptoms in the initial phases, show notable diagnostic difficulties. Their aspecific and late clinical presentation - typical of this site and of the pathology that we are talking about - and the difficult physical-instrumental approach to small bowel limit the possibility of an accurate diagnosis and expose the patient to potentially fatal acute complications and to risks related to emergency surgery treatment that decreases the possibility of a radical resection

Trattamento degli eventi perforativi post-colangiopancreatografia retrograda con sfinterotomia endoscopica. Esperienza personale

Trattamento degli eventi perforativi post-colangio­pancreatografia retrograda con sfinterotomia endoscopica. Esperienza personale. L.G. Angiò, G. Sfuncia, P. Viggiani, G. Faro, A. Bonsignore, M. Licursi, M. Soliera, M. Galati, A. Putortì Introduzione. La colangiopancreatografia retrograda endoscopica (CPRE) ha apportato reali progressi nella gestione delle patologie bilio-pancreatiche, in virtù della sua ambivalenza diagnostica e terapeutica. Le sue complicanze perforative insorgono in meno dell’1% dei pazienti, ma sono associate a una mortalità del 16%-18%. Casistica personale. Caso 1- F, 89 anni con ittero ostruttivo da calcolosi della via biliare principale (VBP) sottoposta a CPRE con sfinterotomia endoscopica (SE), nel corso della quale si verifica lesione tipo II per cui, vista la parziale bonifica della VBP ottenuta, si opta per un trattamento conservativo, con risoluzione in XII giornata post-CPRE. Caso 2- F, 53 anni con colangiti recidivanti e stenosi post-colecistectomia della VBP già trattata mediante stenting; per il verificarsi di lesione tipo I in corso di CPRE, si sottopone la paziente a intervento chirurgico in urgenza/emergenza con guarigione in XXIII giornata. Caso 3- M, 84 anni con colecistite litiasica, ittero ostruttivo, enfisema polmonare e cardiopatia ischemica; eseguita colecistostomia percutanea in urgenza, si procede successivamente a tentativo di CPRE con evidenza di lesione tipo I e, a causa delle comorbilità, si opta per un trattamento conservativo, non risolutivo, per poi procedere all’intervento chirurgico con exitus per complicanze cardio-respiratorie. Caso 4- M, 89 anni con ittero ostruttivo di ndd; interrotta la CPRE per complicanze respiratorie, si procede a colangiografia transeptica per cutanea (CTP) nel corso della quale si evidenzia una lesione tipo IV, che è trattata conservativamente con dimissione in XII giornata. Caso 5- F, 68 anni con colecistite litiasica e coledocolitiasi; in corso di CPRE con SE si verifica lesione tipo II con segni ingravescenti di addome acuto e, in considerazione del quadro clinico e dell’impossibilità di effettuare bonifica endoscopica della VBP, si dispone l’intervento chirurgico in urgenza/emergenza con exitus per complicanze respiratorie. Discussione. Nella diatriba esistente su quale debba essere il trattamento degli eventi perforativi post-CPRE con SE (intervento chirurgico immediato ovvero terapia conservativa), non possiamo non auspicare un atteggiamento eclettico sulla base delle peculiarità anatomo-cliniche del singolo caso