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In vivo expression of an aberrant MYB-GATA1 fusion induces leukemia in the presence of GATA1 reduced levels

Heat shock proteins (HSPs) are a super family of highly conserved molecular chaperone proteins, which are induced in response to stress. HSP70 has been demonstrated to inhibit apoptosis induced by a number of chemotherapeutic agents. Previous investigations have suggested the development of drug resistance in multiple myeloma (MM) cells after adhesion to stroma. This study used MM cell lines and primary plasma cells to determine if HSP70 had a role in development of chemo resistance. Adhesion of MM cells to either bone marrow stromal cells or fibronectin (FN) enhanced HSP70 expression. Inhibition of the HSP70 expression decreased 8226 cell adhesion to stroma or FN and induced more apoptosis in FN-adhered 8226 cells than in suspension cultures at 24 h. Further, HSP70 inhibitors enhanced melphalan-induced apoptosis and reversed melphalan-induced cell adhesion-mediated drug resistance (CAM-DR) phenotype. In addition, compared to parental cells, KNK-437, a heat shock factor inhibitor caused more apoptosis in melphalan-resistant 8226/LR5 cells and sensitized them to melphalan. Primary CD138 positive cells showed high expression of HSPA4 mRNA, and KNK-437 caused apoptosis in these cells. In conclusion, our data suggest inhibition of HSP70, reduced adhesion and caused apoptosis of both acquired and de novo drug resistant MM cells.

A role for bone-marrow-derived cells (BMDCs) in tissue repair and malignancy onset has been proposed, but their contribution is still debated. We tested the ability of BMDCs containing the inducible krasV12 oncogene to initiate lung adenocarcinoma. For our experimental strategy, we reconstituted lethally irradiated wild type mice with BMDCs carrying inducible krasV12 and subsequently induced oncogene expression by 4-OHT administration. Epithelial lung lesions, from adenoma to adenocarcinomas, appeared at successive time points. These results show that lung tumors were derived from donor BMDCs and indicate a direct involvement of bone marrow cells in the development of epithelial cancers.

Despite major advances, multiple myeloma (MM) remains an incurable malignancy. Recently we have found that disease stabilization was achieved in 64% of patients with advanced MM treated with the farnesyltransferase inhibitor R115777 (Zarnestra) in a phase 2 clinical trial. In order to enhance R115777 antitumor activity in MM, we examined the combination of this novel agent with other anticancer drugs in MM cell lines. In this study, R115777 was found to synergize with paclitaxel and docetaxel, but not with other chemotherapy agents, including doxorubicin, 5-fluorouracil, cisplastin, melphalan, mitoxantrone, and dexamethasone. R115777 synergized with paclitaxel to inhibit MM cell proliferation and to induce apoptosis. Synergism in the induction of apoptosis was accompanied by increase in cytochrome c release and caspase-3 activation. Furthermore, flow cytometry analysis also showed that paclitaxel and R115777 synergized to induce G2/M cell-cycle arrest. Importantly, synergism was observed in taxane- and R115777-resistant MM cells. In the human severe combined immunodeficient (SCID-hu) bone model of myeloma growth, the ability of paclitaxel to inhibit tumor growth in vivo was enhanced by R115777. Combination of paclitaxel or docetaxel with R115777 in the treatment of MM cells from patients with multiple myeloma was more beneficial than treatment with single agents. Our results provide the basis for combination therapy clinical trials with paclitaxel or docetaxel with R115777 in MM patients. (Blood. 2005;105:4759-4766)

Patients with multiple myeloma (MM) with mutated RAS are less likely to respond to chemotherapy and have a shortened survival. Therefore, targeting RAS farnesylation may be a novel approach to treatment of MM. We evaluated the activity and tolerability of the farnesyltransferase (FTase) inhibitor tipifarnib (Zarnestra) in a phase 2 trial as well as its ability to inhibit protein farnesylation and oncogenic pathways in patients with relapsed MM. Forty-three patients (median age, 62 years [range, 33-82 years]) with a median of 4 (range, 1-6) chemotherapy regimens entered the study. Tipifarnib, 300 mg orally twice daily, was administered for 3 weeks every 4 weeks. The most common toxicity was fatigue occurring in 66% of patients. Other toxicities included diarrhea, nausea, neuropathy, anemia, and thrombocytopenia. Sixty-four percent of the patients had disease stabilization. Treatment with tipifarnib suppressed FTase (but not geranylgeranyltransferase I) in bone marrow and peripheral blood mononuclear cells and also inhibited the farnesylation of HDJ-2 in unfractionated mononuclear cells and purified myeloma cells. Inhibition of farnesylation did not correlate with disease stabilization. Finally, tipifarnib decreased the levels of phosphorylated Akt and STAT3 (signal transducer and activator of transcription 3) but not Erk1/2 (extracellular signal regulated kinase 1 and 2) in bone marrow cells. We conclude that tipifarnib is tolerable, can induce disease stabilization, and can inhibit farnesylation and oncogenic/tumor survival pathways.

Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are recessive genetic disorders with susceptibility to cancer and similar cellular phenotypes1. The protein product of the gene responsible for A-T, designated ATM, is a member of a family of kinases characterized by a carboxy-terminal phosphatidylinositol 3-kinase-like domain2,3. The NBS1 protein is specifically mutated in patients with Nijmegen breakage syndrome and forms a complex with the DNA repair proteins Rad50 and Mre114,5,6,7. Here we show that phosphorylation of NBS1, induced by ionizing radiation, requires catalytically active ATM. Complexes containing ATM and NBS1 exist in vivo in both untreated cells and cells treated with ionizing radiation. We have identified two residues of NBS1, Ser 278 and Ser 343 that are phosphorylated in vitro by ATM and whose modification in vivo is essential for the cellular response to DNA damage. This response includes S-phase checkpoint activation, formation of the NBS1/Mre11/Rad50 nuclear foci and rescue of hypersensitivity to ionizing radiation. Together, these results demonstrate a biochemical link between cell-cycle checkpoints activated by DNA damage and DNA repair in two genetic diseases with overlapping phenotypes.

A PDGFRB -positive acute myeloid malignancy with a new t(5;12)(q33;p13.3) involving the ERC1 gene

A new dic(7;12)(p12.21;p12.2) chromosome aberration was identified in an acute myeloid leukemia with FAB-M1 morphology and was cloned. Fluorescence in situ hybridization and genomic quantitative polymerase chain reaction mapping experiments revealed the precise localization of the breakpoints, at 7p12, just 5′ to the GRB10 gene, and 12p11, within a genomic region containing no known genes. As a result, a new dicentric chromosome is created, dic(7;12), with the consequent deletion of 50 Mb at 7p, from the telomere to the GRB10 region, and of 30 Mb at 12p, from the telomere to p11. Several genes are included in the affected areas.

The potential of recruited bone-marrow-derived cells (BMDCs) to act as a source of malignancy was disputed for a long time. A role for BMDCs in tissue repair has also been proposed, since these cells are capable of homing to the lung and differentiating into multiple epithelial cell lineages so thus contributing in lung tissue repair and regeneration. We tested the ability of bone marrow cells containing an inducible oncogene to initiate lung adenocarcinoma. Since the role of the K-Ras gene in lung cancer is well known, for our experimental strategy, we used a mouse model expressing oncogenic K-RasV12 (K-Ras+/V12RERTert/ert). This model generates a specific neoplastic phenotype induced by the K-RasV12 mutation. Mice usually develop lung tumors with complete penetrance evolving from hyperplasia, through adenoma, to adenocarcinoma, when the oncogene expression is induced by 4-hydroxitamoxifen (4-OHT) administration. Following lethal irradiation, wild-type mice were transplanted with donor bone marrow cells derived from K-RasV12 mice and, once the bone marrow was reconstituted, treated with 4-OHT. Mice were monitored and sacrificed at subsequent post-induction time points (0, 3.5, 5.5, 8.5, 11, 13.5, 15, and 16 months) and lungs and peripheral blood were collected. Analysis of the H&E-stained lung sections from both reconstituted K-RasV12 and control mice revealed diffuse hyperplasia, possibly due to irradiation. Epithelial lesions, adenomas and/or adenocarcinomas, were detected in 4 out of 8 K-RasV12-reconstituted animals at 3.5, 5.5, 13.5 and 15 months post oncogene induction while no lesions were detected in the control group (transplanted but not induced mice). In addition, the K-Ras implication on the origin of the tumor was confirmed by X-Gal staining of lung lesions in reconstituted mice. In our model, -geo is indeed a marker protein coexpressed along with the same K-RasV12 transcript by a bicistronic strategy. Our results suggest a direct implication of BMDCs in lung epithelial tumor development and emphasize the need of further studies to dissect the mechanisms regulating BMDCs’ recruitment to injured tissues and their role in tumor initiation and/or progression. Citation Format: Elena Belloni, Ines Martin-Padura, Elvira Gerbino, Stefania Orecchioni, Fulvia Fusar-Imperatore, Paola Marighetti, Giovanni Bertalot, Pier Giuseppe Pelicci, Francesco Bertolini. K-RasV12 bone marrow cell reconstitution causes lung cancer in wild type mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2282. doi:10.1158/1538-7445.AM2015-2282

Multiple myeloma (MM) is characterized by the clonal proliferation of malignant plasma cells that accumulate preferentially in the bone marrow. In spite of high dose chemotherapy and novel targeted therapies this disease remains incurable with a median survival of 3–6 years mainly because of the emergence of drug resistance. Improved survival requires new strategies to prevent relapse. Heat shock proteins (HSPs) are a super family of highly conserved proteins, which are induced in plant, yeast, bacterial and mammalian cells in response to an array of physiological and environmental stress cues. Among heat shock protein families, HSP70 is one of the most highly conserved and is the only protein expressed in response to cellular stress. Exogenous HSP70 has been demonstrated to act as a cytokine to human monocytes by stimulating rapid calcium influx, activating nuclear factor (NF)-kB and up-regulating the expression of IL-1b, IL-6 and tumor necrosis factor alpha (TNF-a) (Asea A et al., 2000). Adhesion of myeloma cells to bone marrow stromal cells mediates IL-6 secretion and tumor cell proliferation in part mediated by STAT-3 activation (Cheung WC et al., 2001). We have shown that adhesion of myeloma cells to bone marrow stromal cells enhances IL-6 secretion by stromal cells and HSP70 secretion by myeloma cells. When we inhibited the HSP70 expression using either KNK437 (HSF-1 inhibitor) or RNAi to HSP70, IL-6 secretion by stromal cells as well as activation of STAT-3 in myeloma cells was inhibited in dose-dependent manner. These results suggest that HSP70 released from myeloma cells is enhancing IL-6 secretion from stromal cells. Incubation of stromal cells with recombinant HSP70 did not enhance IL-6 secretion in stromal cells suggesting that some other soluble factor released from myeloma cells cooperates with HSP70 to enhance IL-6 secretion by stromal cells, We examined whether HSP70 can modulate IL-6 mediated STAT-3 activation by stimulating 8226 cells with IL-6 in the presence or absence of KNK437 and RNAi to HSP70 and measuring phospho-STAT-3 by western analysis. HSP70 inhibition attenuated IL-6 induced STAT-3 activity, but not ERK1/2 activity, indicating that HSP70 mediated IL-6 signaling is very specific to STAT-3. The signal transduction cascade by which HSP70 induces IL-6 secretion and the mechanism by which HSP70 mediates IL-6 induced STAT-3 activity are currently under investigation.

Multiple myeloma (MM) is characterized by the clonal proliferation of malignant plasma cells that accumulate preferentially in the bone marrow. In spite of high-dose chemotherapy and novel targeted therapies, myeloma remains to be an incurable disease due to emergence of drug resistance. Therefore, identification of mechanisms involved in drug resistance are essential to develop new and more effective targeted therapies. Heat shock proteins (HSPs) are a super family of highly conserved proteins, which are induced in plant, yeast, bacterial and mammalian cells in response to an array of physiological and environmental stress cues. Among heat shock protein families, HSP70 is one of the most highly conserved and is the only protein expressed in response to cellular stress. HSPs have been implicated in multidrug resistance, as they have been repeatedly demonstrated to inhibit apoptosis induced by a number of chemotherapeutic agents (Chant et al., 1996). We have shown that adhesion of myeloma cells to either bone marrow stromal cells or FN enhances HSP70 expression and secretion as determined by real-time RT-PCR and ELISA, respectively. Inhibition of the HSP70 expression using either KNK437 (HSF-1 inhibitor) or RNAi to HSP70, decreased 8226 cell adhesion to stromal cells as well as to FN as early as two hours, and this adhesion was mediated through α4β1 and α5β1 integrins. Treatment of 8226 cells with KNK437 or RNAi HSP70, induce apoptosis at 24 hours in a dose dependent manner. Interestingly, this effect was independent of adhesion (FN 55% apoptosis vs suspension 42% apoptosis) and is mediated by caspase-3 and PARP cleavage. Further more, treatment of 8226 cells with HSP70 inhibitors reversed CAM-DR to melphalan. To investigate whether HSP70 inhibition can cause apoptosis in Melphalan-resistant myeloma cells, we treated 8226/S and 8226/LR5 cells with either KNK437 alone or in combination with Melphalan. Our results show that KNK437 not only caused more apoptosis in 8226/LR5 (55% with 100 μM) cells than in the sensitive parental cells (42%), but also sensitized 8226/LR5 cells to Melphalan (64%), even though intracellular protein and RNA expression of heat shock protein 27, 70 and 90 was not affected in either Melphalan-sensitive or -resistant cells. These results suggest that 8226/LR5 cells depend on HSP70 for survival more than parental 8226 cells Similarly, pretreatment of 8226 cells with either KNK437, or RNAi against HSP70, enhanced the proteasome inhibitor, Bortezomib- induced apoptosis (Bortezomib 10 nM 8 %, KNK437 25 μM 14 % Combination, 30 %). This apoptosis was mediated by Caspase 3 and was correlated with reduced HSP70 expression. 8226 myeloma cells treated with Bortezomib (10 nM) caused increased RNA and protein expression of HSP70, HSP27 and HSP90 as early as 4 and 8 hrs, respectively. Further studies elucidating the mechanism/s by which HSP70 inhibition sensitizes Melphalan or bortezomib induced apoptosis are currently under investigation. Our preclinical studies provide the basis for potential need for the development of anti HSP70 inhibitors for clinical studies in myeloma.