Thorsten Stühmer

The PI3K/Akt signaling pathway regulates the expression of Hsp70, which critically contributes to Hsp90-chaperone function and tumor cell survival in multiple myeloma

Despite therapeutic advances multiple myeloma remains largely incurable, and novel therapeutic concepts are needed. The Hsp90-chaperone is a reasonable therapeutic target, because it maintains oncogenic signaling of multiple deregulated pathways. However, in contrast to promising preclinical results, only limited clinical efficacy has been achieved through pharmacological Hsp90 inhibition. Because Hsp70 has been described to interact functionally with the Hsp90-complex, we analyzed the suitability of Hsp72 and Hsp73 as potential additional target sites. Expression of Hsp72 and Hsp73 in myeloma cells was analyzed by immunohistochemical staining and western blotting. Short interfering RNA-mediated knockdown or pharmacological inhibition of Hsp72 and Hsp73 was performed to evaluate the role of these proteins in myeloma cell survival and for Hsp90-chaperone function. Furthermore, the role of PI3K-dependent signaling in constitutive and inducible Hsp70 expression was investigated using short interfering RNA-mediated and pharmacological PI3K inhibition. Hsp72 and Hsp73 were frequently overexpressed in multiple myeloma. Knockdown of Hsp72 and/or Hsp73 or treatment with VER-155008 induced apoptosis of myeloma cells. Hsp72/Hsp73 inhibition decreased protein levels of Hsp90-chaperone clients affecting multiple oncogenic signaling pathways, and acted synergistically with the Hsp90 inhibitor NVP-AUY922 in the induction of death of myeloma cells. Inhibition of the PI3K/Akt/GSK3β pathway with short interfering RNA or PI103 decreased expression of the heat shock transcription factor 1 and down-regulated constitutive and inducible Hsp70 expression. Treatment of myeloma cells with a combination of NVP-AUY922 and PI103 resulted in additive to synergistic cytotoxicity. In conclusion, Hsp72 and Hsp73 sustain Hsp90-chaperone function and critically contribute to the survival of myeloma cells. Translation of Hsp70 inhibition into the clinic is therefore highly desirable. Treatment with PI3K inhibitors might represent an alternative therapeutic strategy to target Hsp70.

Signalling profile and antitumour activity of the novel Hsp90 inhibitor NVP-AUY922 in multiple myeloma.

We as well as others have recently shown that Hsp90 is overexpressed in multiple myeloma (MM) and critically contributes to tumour cell survival. Pharmacologic blockade of Hsp90 has consistently been found to induce MM cell death. However, most data have been obtained with MM cell lines whereas knowledge about the molecular effects of pharmacologic Hsp90 blockade in primary tumour cells is limited. Furthermore, these investigations have so far focused on geldanamycin derivatives. We analysed the biochemical effects of a novel diarylisoxazole-based Hsp90 inhibitor (NVP-AUY922) on signalling pathways and cell death in a large set of primary MM tumour samples and in MM cell lines. Treated cells displayed the molecular signature and pharmacodynamic properties for abrogation of Hsp90 function, such as downregulation of multiple survival pathways and strong upregulation of Hsp70. NVP-AUY922 treatment efficiently induced MM cell apoptosis and revealed both sensitive and resistant subgroups. Sensitivity was not correlated with TP53 mutation or Hsp70 induction levels and stromal cells from the bone marrow microenvironment were unable to abrogate NVP-AUY922-induced apoptosis of MM cells. Thus, NVP-AUY922 may be a promising drug for treatment of MM and clinical studies are warranted.

Combined functional and molecular analysis of tumor cell signaling defines 2 distinct myeloma subgroups: Akt-dependent and Akt-independent multiple myeloma

Although the phosphatidylinositide 3-kinase (PI3K)/Akt pathway has been reported to contribute to the malignant growth of multiple myeloma (MM), the true relevance of Akt kinases for this disease is still unclear. In particular, functional analyses in primary tumor cells and genetic target validation experiments are missing. Here, we used combined functional and molecular analyses to determine the importance of Akt activity in a large panel of primary MM samples and in MM cell lines. Akt down-regulation with isoform-specific siRNA constructs or with an Akt1/2-specific pharmacologic inhibitor strongly induced apoptosis in approximately half of the primary MM samples analyzed. Sensitivity to Akt inhibition strongly correlated with the activation status of Akt as determined by immunohistochemistry, phospho-Akt-specific flow cytometry, and Western analysis. Additional blockade of the MAPK and the IL-6R/STAT3 pathways was often not sufficient to decrease the viability of MM cells resilient to Akt inhibition. Taken together, these experiments led to the identification of 2 myeloma subgroups: Akt-dependent and Akt-independent MM.

The heat shock transcription factor 1 as a potential new therapeutic target in multiple myeloma

The heat shock transcription factor 1 (HSF1) has recently been reported to promote malignant transformation and growth. Here we provide experimental evidence for a role of HSF1 in the pathogenesis of multiple myeloma (MM). Immunohistochemical analyses revealed that HSF1 was overexpressed in half of the investigated MM samples, including virtually all cases with extramedullary manifestations or anaplastic morphology. HSF1 function was inhibited either by siRNA‐mediated knockdown or pharmacologically through treatment with triptolide. Both approaches caused depletion of HSF1, lowered the constitutively high expression of a multitude of protective HSPs (such as HSP90, HSP70, HSP40 and HSP27), induced apoptosis in human MM cells in vitro, and strongly reduced MM tumour growth in vivo. Furthermore, we observed that treatment‐induced upregulation of HSPs after proteasome or HSP90 inhibition was critically dependent on HSF1. Importantly, the apoptotic effects of the HSP90 inhibitor NVP‐AUY922 or the proteasome inhibitor bortezomib were strongly enhanced in combination with triptolide, suggesting a salvage role of HSF1‐dependent HSP induction in response to drug treatment. Collectively, our data indicate that inhibition of HSF1 affects multiple protective HSPs and might therefore represent a therapeutic strategy – in particular in combination with proteasome or HSP90 inhibitors.

Preclinical anti-myeloma activity of the novel HDAC-inhibitor JNJ-26481585

Pharmacological inhibitors of histone deacetylases (HDACs) are currently being developed and tested as anti‐cancer agents and may be useful to enhance the therapeutic efficiency of established anti‐myeloma treatments. This study preclinically evaluated the effects of the ‘second generation’ pan‐HDAC inhibitor JNJ‐26481585 on human multiple myeloma (MM) cells from established cell lines and primary MM samples (n = 42). Molecular responses in both groups of MM cells included histone acetylation, a shift in Bcl2‐family members towards proapoptotic bias, attenuation of growth and survival pathway activity and Hsp72 induction. Mcl‐1 depletion and Hsp72 induction were the most reliable features observed in JNJ‐26481585‐treated primary MM samples. The drug alone effectively induced myeloma cell death at low nanomolar concentrations. In vitro combination of JNJ‐26481585 with anti‐myeloma therapeutic agents generally resulted In effects close to additivity. In view of the favourable activity of this novel HDAC‐inhibitor towards primary myeloma cells further evaluation in a clinical setting is warranted.

Pharmacologic activation of p53-dependent and p53-independent apoptotic pathways in Hodgkin/Reed-Sternberg cells

The status of the p53 pathway in classical Hodgkin lymphoma (cHL) remains unclear, and a lack of proven TP53 mutations contrasts with often high expression levels of p53 protein. In this study, we demonstrate that pharmacologic activation of the p53 pathway with the murine double minute 2 (MDM2) antagonist nutlin-3 in Hodgkin lymphoma-derived cell lines leads to effective apoptosis induction and sensitizes the cells to other anticancer drugs. Cells with mutant p53 are resistant to nutlin-3, but sensitive to geldanamycin, a pharmacologic inhibitor of heat shock 90 kDa protein (HSP90), indicating that HSP90 inhibition can induce apoptosis in a p53-independent manner. Conversely, cells with defects in the HSP90/nuclear factor-κ B pathway expressing wild-type p53 are more resistant to geldanamycin, but still sensitive to nutlin-3. Our results suggest that selective activation of p53 by MDM2 antagonists as a single agent or in combination with conventional chemotherapeutics and/or inhibitors of p53-independent survival pathways may offer effective treatment options for patients with cHL. Importantly, because nutlins and HSP90 inhibitors are non-genotoxic agents, their use might offer a means to reduce the genotoxic burden of current chemotherapeutic regimens.

Combined targeting of MEK/MAPK and PI3K/Akt signalling in multiple myeloma

So‐called RAS‐dependent pathways, such as those signalling via mitogen‐activated protein kinase kinase (MEK)/mitogen‐activated protein kinase (MAPK) and phosphoinositide‐3 kinase (PI3K)/Akt, are implicated in proliferation and survival of multiple myeloma (MM) cells. However, the effects of their combined blockade and its potential therapeutic utility for the treatment of RAS‐mutated MM have not systematically been analysed. Here, we tested the functional consequences of single versus combined inhibition of the MEK/MAPK and PI3K/Akt pathways in a large series of primary MM samples (n = 55) and MM cell lines (n = 11). Additionally, the anti‐myeloma activity of different treatments was analysed with respect to the RAS mutation status. PI3K/Akt blockade was generally more pro‐apoptotic than blockade of MEK/MAPK both in cell lines and in primary MM samples. Simultaneous blockade of both pathways led to significantly enhanced anti‐myeloma activity in 75% of primary MM samples, whereas the remainder was largely resistant. Resistance to combination blockade was exclusively observed in RAS wildtype cases, whereas sensitivity was noted in RAS wildtype and in RAS mutated MM. These results suggest that oncogenic RAS is a predictor of sensitivity to combination treatment with PI3K/Akt and MEK/MAPK inhibitors and that such an approach might therefore be beneficial for this genetically well‐defined subgroup of MM patients.

Antitumoral and antileishmanial dioncoquinones and ancistroquinones from cell cultures of Triphyophyllum peltatum (Dioncophyllaceae) and Ancistrocladus abbreviatus (Ancistrocladaceae)

From the methanolic extracts of solid callus cultures from two species of the closely related palaeotropical plant families Dioncophyllaceae and Ancistrocladaceae seven new natural naphthoquinones were isolated, dioncoquinones A (4) and B (5) from Triphyophyllum peltatum, and ancistroquinones B (6), C (7), D (9), E (10), and F (12) from Ancistrocladus abbreviatus. Their structures were elucidated by spectroscopic, chemical, and computational methods. Furthermore, the already known naphthoquinones plumbagin (2), droserone (3), malvone A (8), and nepenthone A (11) were found in the extract of A. abbreviatus. Dioncoquinones A (4) and B (5) showed good - and specific - activity against Leishmania major, while they were not active against other protozoic parasites. Moreover, treatment with 4 and 5 strongly induced apoptosis in human tumor cells derived from two different B cell malignancies, B cell lymphoma and multiple myeloma, without any significant toxicity towards normal peripheral mononuclear blood cells.

Multiple myeloma is affected by multiple and heterogeneous somatic mutations in adhesion- and receptor tyrosine kinase signaling molecules

Multiple myeloma (MM) is a largely incurable plasma cell malignancy with a poorly understood and heterogeneous clinical course. To identify potential, functionally relevant somatic mutations in MM, we performed whole-exome sequencing of five primary MM, corresponding germline DNA and six MM cell lines, and developed a bioinformatics strategy that also integrated published mutational data of 38 MM patients. Our analysis confirms that identical, recurrent mutations of single genes are infrequent in MM, but highlights that mutations cluster in important cellular pathways. Specifically, we show enrichment of mutations in adhesion molecules of MM cells, emphasizing the important role for the interaction of the MM cells with their microenvironment. We describe an increased rate of mutations in receptor tyrosine kinases (RTKs) and associated signaling effectors, for example, in EGFR, ERBB3, KRAS and MAP2K2, pointing to a role of aberrant RTK signaling in the development or progression of MM. The diversity of mutations affecting different nodes of a particular signaling network appears to be an intrinsic feature of individual MM samples, and the elucidation of intra- as well as interindividual redundancy in mutations that affect survival pathways will help to better tailor targeted therapeutic strategies to the specific needs of the MM patient.

Oligomerized tumor necrosis factor‐related apoptosis inducing ligand strongly induces cell death in myeloma cells, but also activates proinflammatory signaling pathways

The oligomerization status of soluble tumor necrosis factor‐related apoptosis inducing ligand (TRAIL) trimers has an overwhelming impact on cell death induction in a cell‐type dependent fashion. Thus, we evaluated the ability of single and oligomerized TRAIL trimers to induce cell death in human myeloma cells. In all myeloma cell lines analyzed, oligomerized TRAIL trimers induced caspase activation and complete cell death, whereas non‐oligomerized TRAIL trimers showed no or only a modest effect. Caspase activation induced by oligomerized TRAIL was blocked in all cell lines by the pan‐caspase inhibitor benzyloxycarbonyl‐Val‐Ala‐Asp(OMe)‐fluoromethylketone (z‐VAD‐fmk). Cell death induction was largely blocked in two cell lines by z‐VAD‐fmk, but was only marginally attenuated in three other cell lines, indicating that TRAIL induces caspase‐dependent and caspase‐independent cell death in myeloma cells. Preceding cell death, TRAIL activated nuclear factor κB, c‐Jun N‐terminal kinase, p38 and p42/44. Although TRAIL‐induced stimulation of c‐Jun N‐terminal kinase and p38 was caspase‐dependent in a cell type‐specific fashion, activation of nuclear factor κB and p42/44 was caspase‐independent in all cases. In accordance with activation of the nuclear factor κB pathway, we observed transcriptional up‐regulation of several well established nuclear factor κB target genes. Furthermore, we found that TRAIL activates proinflammatory pathways in approximately 50% of primary myeloma samples. Taken together, our data suggest (a) that oligomerized TRAIL variants are necessary to ensure maximal cell death induction in myeloma cells and (b) TRAIL should be used in combination with anti‐inflammatory drugs for treatment of myeloma to avoid and/or minimize any potential side‐effects arising from the proinflammatory properties of the molecule.