Project description:Our previous studies have shown that inhibition of the IGF-1R pathway by the IGF-1RTK inhibitor picropodophyllin (PPP) can be achieved and also constitutes a favorable therapeutic window in multiple myeloma (MM). As no complete remission using in vivo models of MM could be obtained, a combinatorial drug screen (HTS) was performed to select the most performant combination with PPP. The HDAC inhibitor LBH589 was shown to act in synergy with PPP on survival of MM cells. The contribution from both drugs and the combination were further monitored for apoptosis, cell cycle distribution, and the impact on downstream gene and protein expression in human and mouse MM models in vitro. In the RPMI 8226 human MM cell line, simultaneous treatment with both compounds for 48h caused a 5-fold increase of apoptotic and late apoptotic/necrotic cells as compared to controls, while treatment with either compound alone only induced a 3-fold increase. After 24h cleavage of apoptotic proteins caspase -9, -8 and -3 could be found in RPMI 8226 cells treated with both drugs individually, but in the combination we observed an additive effect on the cleavage of the active forms of caspase 8 as compared to single drug treatments. The combination of LBH589 and PPP could be monitored as an accumulation of cells in the G2/M phase, and subsequent down-regulation of cell cycle regulated proteins. The effect of both compounds on the expression of cyclin B1, -E and -D2 was additive, as demonstrated by western blot. These data were also confirmed in the mouse 5T33MM cells in vitro. Gene expression analysis and validations of the RPMI 8226 cells reveal that the drug combination has better effects than the single drug alone. Combined treatment in vivo resulted in a significant prolonged survival of 5T33MM inoculated mice when compared to the control group and to treatment with the drugs alone. In conclusion, the results indicate an improved MM treatment opportunity in using a combination of PPP and LBH589. We used whole genome Microarray to decipher the changes occuring after treatment of the single and combination of the drugs. And see if the combination has better effects.

Project description:Our previous studies have shown that inhibition of the IGF-1R pathway by the IGF-1RTK inhibitor picropodophyllin (PPP) can be achieved and also constitutes a favorable therapeutic window in multiple myeloma (MM). As no complete remission using in vivo models of MM could be obtained, a combinatorial drug screen (HTS) was performed to select the most performant combination with PPP. The HDAC inhibitor LBH589 was shown to act in synergy with PPP on survival of MM cells. The contribution from both drugs and the combination were further monitored for apoptosis, cell cycle distribution, and the impact on downstream gene and protein expression in human and mouse MM models in vitro. In the RPMI 8226 human MM cell line, simultaneous treatment with both compounds for 48h caused a 5-fold increase of apoptotic and late apoptotic/necrotic cells as compared to controls, while treatment with either compound alone only induced a 3-fold increase. After 24h cleavage of apoptotic proteins caspase -9, -8 and -3 could be found in RPMI 8226 cells treated with both drugs individually, but in the combination we observed an additive effect on the cleavage of the active forms of caspase 8 as compared to single drug treatments. The combination of LBH589 and PPP could be monitored as an accumulation of cells in the G2/M phase, and subsequent down-regulation of cell cycle regulated proteins. The effect of both compounds on the expression of cyclin B1, -E and -D2 was additive, as demonstrated by western blot. These data were also confirmed in the mouse 5T33MM cells in vitro. Gene expression analysis and validations of the RPMI 8226 cells reveal that the drug combination has better effects than the single drug alone. Combined treatment in vivo resulted in a significant prolonged survival of 5T33MM inoculated mice when compared to the control group and to treatment with the drugs alone. In conclusion, the results indicate an improved MM treatment opportunity in using a combination of PPP and LBH589. We used whole genome Microarray to decipher the changes occuring after treatment of the single and combination of the drugs. And see if the combination has better effects. RPMI 8266 cells were treated with LBH589 (20nM), picropodophyllin (PPP)(0.375uM) , and the combination of these two drugs. RNA was extracted at two different time points 6 and 24 for the microarray.

Project description:Natural Killer (NK) cells act as important regulators in the development and progression of hematological malignancies and their suppressor activity against Multiple Myeloma (MM) cells has been confirmed in many studies. Significant changes in the distribution of NK cell subsets and dysfunctions of NK cell effector activities were described in MM patients and correlated with disease staging. Thus, restoring or enhancing the functionality of these effectors for the treatment of MM represents a critical need. Neddylation is a post-translational modification that adds a ubiquitin-like molecule, NEDD8, to the substrate protein. One of the outcomes is the activation of the Cullin Ring Ligases (CRLs), a class of ubiquitin-ligases that controls the degradation of about 20% of proteasome-regulated proteins. Overactivation of CRLs has been described in cancer and can lead to tumor growth and progression. Thus, targeting neddylation represents an attractive approach for cancer treatment. Our group has recently described how pharmacologic inhibition of neddylation increases the expression of the NKG2D activating receptor ligands, MICA and MICB, in MM cells, making these cells more susceptible to NK cell degranulation and killing. Here, we extended our investigation to the direct role of neddylation on NK cell effector functions exerted against MM. We observed that inhibition of neddylation enhanced NK cell-mediated degranulation and killing against MM cells and improved Daratumumab/Elotuzumab-mediated response. Mechanistically, inhibition of neddylation increased the expression of Rac1 and RhoA GTPases in NK cells, critical mediators for an efficient degranulation at the immunological synapse of cytotoxic lymphocytes, and augmented the levels of F-actin and perforin polarization in NK cells contacting target cells. Moreover, inhibition of neddylation partially abrogated TGFβ-mediated repression of NK cell effector activity. This study describes the role of neddylation on NK cell effector functions and highlights the positive immunomodulatory effects achieved by the inhibition of this pathway in MM.

Project description:BackgroundMechanisms by which Smac mimetics (SMs) interact with proteasome inhibitors (e.g., bortezomib) are largely unknown, particularly in multiple myeloma (MM), a disease in which bortezomib represents a mainstay of therapy.MethodsInteractions between the clinically relevant IAP (inhibitor of apoptosis protein) antagonist birinapant (TL32711) and the proteasome inhibitor bortezomib were investigated in multiple myeloma (MM) cell lines and primary cells, as well as in vivo models. Induction of apoptosis and changes in gene and protein expression were monitored using MM cell lines and confirmed in primary MM cell populations. Genetically modified cells (e.g., exhibiting shRNA knockdown or ectopic expression) were employed to evaluate the functional significance of birinapant/bortezomib-induced changes in protein levels. A MM xenograft model was used to evaluate the in vivo activity of the birinapant/bortezomib regimen.ResultsBirinapant and bortezomib synergistically induced apoptosis in diverse cell lines, including bortezomib-resistant cells (PS-R). The regimen robustly downregulated cIAP1/2 but not the canonical NF-κB pathway, reflected by p65 phosphorylation and nuclear accumulation. In contrast, the bortezomib/birinapant regimen upregulated TRAF3, downregulated TRAF2, and diminished p52 processing and BCL-XL expression, consistent with disruption of the non-canonical NF-κB pathway. TRAF3 knockdown, ectopic TRAF2, or BCL-XL expression significantly diminished birinapant/bortezomib toxicity. The regimen sharply increased extrinsic apoptotic pathway activation, and cells expressing dominant-negative FADD or caspase-8 displayed markedly reduced birinapant/bortezomib sensitivity. Primary CD138+ (n = 43) and primitive MM populations (CD138-/19+/20+/27+; n = 31) but not normal CD34+ cells exhibited significantly enhanced toxicity with combined treatment (P < 0.0001). The regimen was also fully active in the presence of HS-5 stromal cells or growth factors (e.g., IL-6 and VEGF). Finally, the regimen was well tolerated and significantly increased survival (P < 0.05 and P < 0.001) compared to single agents in a MM xenograft model. Combined treatment also downregulated cIAP1/2 and p52 while increasing PARP cleavage in MM cells in vivo.ConclusionsOur data suggest that birinapant and bortezomib interact synergistically in MM cells, including those resistant to bortezomib, through inactivation of the non-canonical NF-κB and activation of the extrinsic apoptotic pathway both in vitro and in vivo. They also argue that a strategy combining cIAP antagonists and proteasome inhibitors warrants attention in MM.

Project description:Many types of cancer such as prostate cancer, myeloid leukemia, breast cancer, glioblastoma display strong chemo resistance, which is supported by enhanced expression of multiple anti-apoptotic Bcl-2, Bcl-XL and Mcl-1 proteins. The viable anti-cancer strategies are based on developing anti-apoptotic Bcl-2 proteins inhibitors, BH3 mimetics. Our focus in past years has been on the investigating a new potential BH3 mimetic, Hypericin (Hyp). Hyp is a naturally occurring photosensitive compound used in photodynamic therapy and diagnosis. We have demonstrated that Hyp can cause substantial effects in cellular ultrastructure, mitochondria function and metabolism, and distribution of Bcl2 proteins in malignant and non-malignant cells. One of the possible mechanisms of Hyp action could be the direct interactions between Bcl-2 proteins and Hyp. We investigated this assumption by in silico computer modelling and in vitro fluorescent spectroscopy experiments with the small Bcl2 peptide segments designed to correspond to Bcl2 BH3 and BH1 domains. We show here that Hyp interacts with BH3 and BH1 peptides in concentration dependent manner, and shows the stronger interactions than known BH3 mimetics, Gossypol (Goss) and ABT-263. In addition, interactions of Hyp, Goss and ABT263, with whole purified proteins Bcl-2 and Mcl-1 by fluorescence spectroscopy show that Hyp interacts stronger with the Bcl-2 and less with Mcl-1 protein than Goss or ABT-263. This suggest that Hyp is comparable to other BH3 mimetics and could be explore as such. Hyp cytotoxicity was low in human U87 MG glioma, similar to that of ABT263, where Goss exerted sufficient cytotoxicity, suggesting that Hyp acts primarily on Bcl-2, but not on Mcl-1 protein. In combination therapy, low doses of Hyp with Goss effectively decreased U87 MG viability, suggesting a possible synergy effect. Overall, we can conclude that Hyp as BH3 mimetic acts primarily on Bcl-2 protein and can be explored to target cells with Bcl-2 over-expression, or in combination with other BH3 mimetics, that target Mcl-1 or Bcl-XL proteins, in dual therapy.

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The IGF-1 receptor inhibitor picropodophyllin potentiates the anti-myeloma activity of a BH3-mimetic.

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The IGF-1 receptor inhibitor picropodophyllin potentiates the anti-myeloma activity of a BH3-mimetic.

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