Drug resistance in multiple myeloma associated with high in vitro incorporation of 3H-thymidine

In multiple myeloma, tumor cell mass and labeling index correlate with subsequent survival duration, but do not predict for response to treatment. In the present study was have autoradiographically measured the incorporation of 3H-thymidine as determined by the number of grains over the myeloma nuclei in bone marrow aspiration samples. In 33/37 patients with less than 50% tumor regression or progressive disease, the pretreatment grain count was greater than or equal to 20/myeloma nucleus. Conversely, values of less than 20 were found in 27/29 patients who had greater than or equal to 50% cell mass reduction. Survival duration was significantly better than (p less than 0.001) in patients with grain counts less than 20. Sixty percent of the patients with both a low labeling index (less than or equal to 3%) and grain count (less than 20) were alive at 48 mo, whereas 15/17 patients with a high labeling index and grain count had a median survival of less than 6 mo. In a subset of 22 patients, there as a significant correlation between in vitro resistance to melphalan, adriamycin, and vincristine as tested in the myeloma stem cell colony assay system and a grain count of greater than 20. We can only speculate as to the reasons for the increased 3H-thymidine uptake by myeloma cells resistant to treatment, however, it could be associated with accumulation of excess DNA and /or increased unscheduled DNA synthesis following injury from alkylating agents.

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Drug resistance in multiple myeloma associated with high in vitro incorporation of 3H-thymidine

Blood ◽

10.1182/blood.v58.3.471.471 ◽

1981 ◽

Vol 58 (3) ◽

pp. 471-476 ◽

Cited By ~ 24

Author(s):

V Hofmann ◽

SE Salmon ◽

BG Durie

Keyword(s):

Multiple Myeloma ◽

Tumor Regression ◽

Alkylating Agents ◽

Cell Mass ◽

Bone Marrow Aspiration ◽

Labeling Index ◽

Response To Treatment ◽

Survival Duration ◽

Thymidine Uptake

Abstract In multiple myeloma, tumor cell mass and labeling index correlate with subsequent survival duration, but do not predict for response to treatment. In the present study was have autoradiographically measured the incorporation of 3H-thymidine as determined by the number of grains over the myeloma nuclei in bone marrow aspiration samples. In 33/37 patients with less than 50% tumor regression or progressive disease, the pretreatment grain count was greater than or equal to 20/myeloma nucleus. Conversely, values of less than 20 were found in 27/29 patients who had greater than or equal to 50% cell mass reduction. Survival duration was significantly better than (p less than 0.001) in patients with grain counts less than 20. Sixty percent of the patients with both a low labeling index (less than or equal to 3%) and grain count (less than 20) were alive at 48 mo, whereas 15/17 patients with a high labeling index and grain count had a median survival of less than 6 mo. In a subset of 22 patients, there as a significant correlation between in vitro resistance to melphalan, adriamycin, and vincristine as tested in the myeloma stem cell colony assay system and a grain count of greater than 20. We can only speculate as to the reasons for the increased 3H-thymidine uptake by myeloma cells resistant to treatment, however, it could be associated with accumulation of excess DNA and /or increased unscheduled DNA synthesis following injury from alkylating agents.

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Engineering VSV-IFN-NIS for the Treatment of Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.378.378 ◽

2009 ◽

Vol 114 (22) ◽

pp. 378-378

Author(s):

Shruthi Naik ◽

Rebecca A. Nace ◽

Elizabeth A. Hadac ◽

David Dingli ◽

Mark Federspiel ◽

...

Keyword(s):

Multiple Myeloma ◽

Conflicts Of Interest ◽

Tumor Regression ◽

Oncolytic Viruses ◽

Sodium Iodide Symporter ◽

Viral Spread ◽

Nis Gene ◽

Immunocompetent Mice

Abstract Abstract 378 The need for new and more effective long-term treatments for Multiple Myeloma (MM) has led to the utilization and engineering of replication competent (oncolytic) viruses as potential therapies. Vesicular stomatitis virus (VSV) is a potent oncolytic agent with several features that make it a favorable choice as a potential myeloma therapy. Specifically, (i) VSV replicates rapidly and can be grown to high titers (for effective clinical use) (ii) VSV undergoes transcription and replication exclusively in the cytoplasm avoiding host genome integration (iii) there is low/absent pre-existing immunity against VSV among the general population, (iv) naturally occurring human VSV infections are generally asymptomatic or result in minimal flu-like symptoms, (v) VSV is not easily transmitted between individuals (natural transmission is by hematophagous insects). Previously we showed weak oncolytic efficacy with an attenuated strain of VSV coding for the sodium iodide symporter (NIS) gene, VSV-D51-NIS, in the immune competent 5TGM1 syngeneic MM mouse model (C57Bl/KalwRijHsd) (Goel at. al. Blood. 2007 Oct 1;110(7):2342-50). Since VSV replication is strongly inhibited by IFN-induced innate immune responses in normal cells, but not in myeloma cells and IFN has known anti-myeloma activity, we hypothesized that the IFN-coding VSVs would be safer and more potent than previously tested VSV recombinants. We therefore constructed and tested the efficacy of VSVs coding for b-Interferon (VSV-IFN) or b-IFN and NIS (VSV-IFN-NIS). Interestingly, all of the newly constructed viruses, including VSV-IFN-NIS, showed greatly superior replication kinetics compared to the previously reported VSV-D51-NIS virus. Furthermore, compared to VSV-D51-NIS, VSV-IFN-NIS vectors induced higher NIS expression in vitro. In vivo therapy studies showed that a single intravenous dose of each of the IFN-coding VSVs promoted tumor regression and significantly prolonged survival of immunocompetent mice bearing subcutaneous or orthotopic 5TGM1 myeloma tumors. Tc-99m imaging studies conducted in mice treated with VSV-IFN-NIS, showed tumor specific virus mediated NIS expression and radio-isotope uptake that increased concurrently with intratumoral viral spread. Most importantly, there was no evidence of neurotoxicity following treatment with the IFN-coding VSVs. These studies indicate that VSV-IFN-NIS has potential as a novel therapeutic agent for multiple myeloma that can be combined with radio-isotopes for both non-invasive imaging of viral biodistribution and radiovirotherapy. A phase I clinical study is currently planned. Disclosures: No relevant conflicts of interest to declare.

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PKC412 Is a Multi-Targeting Kinase Inhibitor with Activity Against Multiple Myeloma In Vitro and In Vivo.

Blood ◽

10.1182/blood.v106.11.247.247 ◽

2005 ◽

Vol 106 (11) ◽

pp. 247-247 ◽

Cited By ~ 2

Author(s):

Joseph Negri ◽

Nicholas Mitsiades ◽

Qingwei Deng ◽

Zhaoqin Wen ◽

David C. Geer ◽

...

Keyword(s):

Multiple Myeloma ◽

Tyrosine Kinase ◽

Cell Lines ◽

Kinase Inhibitor ◽

De Novo ◽

Alkylating Agents ◽

Nod Mice ◽

Protective Effects

Abstract Multiple myeloma (MM) remains an incurable neoplasia and exhibits high propensity for de novo/acquired refractoriness even to novel agents, e.g. thalidomide (Thal) or proteasome inhibitors. This may be due to complex and evolving patterns of molecular lesions potentially conferring hyperactive antiapoptotic signaling with high degree of redundancy upon inhibition of isolated targets within those pathways. We thus hypothesized that, for genetically complex neoplasias such as MM, patient outcome might improved by addition, in the therapeutic armamentarium, of agents that simultaneously inhibit multiple proliferative/antiapoptotic targets. Towards this effort of multi-targeted therapies, we studied the tyrosine kinase inhibitor PKC412 (midausporin, Novartis, Basel, Switzerland). Low-nM levels of PKC412 selectively inhibit tyrosine kinase receptors, such as FLT3. But higher PKC412 concentrations (in μM range), which presumably inhibit (at least partly) other kinases, are achieved in clinical trials without catastrophic toxicities. This suggests that PKC412 can safely suppress in vivo the activity of, not just FLT3, but a broader spectrum of kinases, some of which (individually or cooperatively) might be critical for survival/proliferation of MM cells. Indeed, in vitro kinase activity assays showed that clinically achievable (high nM or low μM) PKC412 concentrations inhibit by >20% important kinases, including p70S6K, IKK-a and Akt,. Consistent with such multi-targeted effect, in vitro MTT colorimetric survival assays showed activity of PKC412 (at sub-μM levels) against panel of 40 MM cell lines and 10 primary tumor cells from MM patients (including cells resistant to Dex, alkylating agents, anthracyclines, Thal or its immunomodulatory derivatives, bortezomib, and/or Apo2L/TRAIL), and cell lines from hematologic neoplasias, e.g. B- and T-ALL, CML, various non-Hodgkin’s lymphoma subtypes, and solid tumors (e.g. breast, prostate, lung, thyroid, ovarian, renal Ca, retinoblastoma and sarcomas)). Mechanistic studies confirmed that PKC412 blocks key growth/survival pathways (e.g. PI-3K/Akt, IKK-α/NF-κB), coupled with by downstream effects on suppression of diverse inhibitors of apoptosis (e.g. FLIP, XIAP, cIAP-2, survivin). These molecular sequelae explain, at least partly, the ability of PKC412 to sensitize MM cells to other anti-MM agents (such as Dex, cytotoxic chemotherapy or proteasome inhibitor bortezomib) and overcome protective effects of cytokines (e.g. IL-6) or bone marrow stromal cells. Importantly, PKC412 significantly prolonged the overall survival (p<0.03, Kaplan-Meier analysis) of SCID/NOD mice in a clinically relevant model of diffuse MM bones lesions. These studies provide basis for clinical studies of PKC412 in MM and indicate that kinase inhibitors selectively blocking specific targets at low drug levels, may also have potent anti-tumor activities related to inhibition of multiple other, less specific, nonetheless important targets, thus allowing for anti-tumor activity in a much broader spectrum of tumor types than previously appreciated.

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Plasma cell acid phosphatase activity as prognostic factor in multiple myeloma: relationship to the thymidine-labeling index.

Journal of Clinical Oncology ◽

10.1200/jco.1985.3.11.1503 ◽

1985 ◽

Vol 3 (11) ◽

pp. 1503-1507 ◽

Cited By ~ 4

Author(s):

M Boccadoro ◽

A Gallamini ◽

A Fruttero ◽

P Gavarotti ◽

V Redoglia ◽

...

Keyword(s):

Multiple Myeloma ◽

Acid Phosphatase ◽

Prognostic Factor ◽

Plasma Cell ◽

Patient Survival ◽

Labeling Index ◽

Survival Duration ◽

Monoclonal Gammopathies ◽

Thymidine Labeling Index ◽

Cell Acid

Plasma cell acid phosphatase (AP) activity and thymidine labeling index (LI%) were evaluated concomitantly in 52 patients with monoclonal gammopathies. AP score, percentage of AP positive plasma cells, and LI% were significantly higher in 26 patients with multiple myeloma (MM) at the time of diagnosis than in 11 monoclonal gammopathy of undetermined significance (MGUS) and eight smoldering myeloma (SM) patients. LI% had the highest statistical correlation with disease status. A 1% cutoff could clearly separate the patients with progressive MM compared to those with stable disease (SM-MGUS) (P less than .001). There was a significant overall correlation between the AP score and LI% (P less than .005). Since LI% is a recognized powerful prognostic factor, this correlation suggests that the AP score can also be a reliable test predicting patient survival duration. In addition, we identified a subgroup of IgG MM patients with very high tumor mass who had a low LI% but a high AP score. This was associated with very poor patient survival and indicated the discrete prognostic importance of AP score in this subgroup with low LI%. Thus, both the LI% and AP score can be recommended as helpful clinical tests in patients with monoclonal gammopathies.

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A Novel Antibody-Drug Conjugate (ADC) Delivering a DNA Mono-Alkylating Payload to Chondroitin Sulfate Proteoglycan (CSPG4)-Expressing Melanoma

Cancers ◽

10.3390/cancers12041029 ◽

2020 ◽

Vol 12 (4) ◽

pp. 1029

Author(s):

Ricarda M. Hoffmann ◽

Silvia Crescioli ◽

Silvia Mele ◽

Eirini Sachouli ◽

Anthony Cheung ◽

...

Keyword(s):

Tumor Regression ◽

Chondroitin Sulphate ◽

Alkylating Agents ◽

Residual Tumor ◽

Similar Efficacy ◽

Antibody Drug Conjugate ◽

Drug Conjugates ◽

Antibody Drug

Despite emerging targeted and immunotherapy treatments, no monoclonal antibodies or antibody-drug conjugates (ADCs) directly targeting tumor cells are currently approved for melanoma therapy. The tumor-associated antigen chondroitin sulphate proteoglycan 4 (CSPG4), a neural crest glycoprotein over-expressed on 70% of melanomas, contributes to proliferative signaling pathways, but despite highly tumor-selective expression it has not yet been targeted using ADCs. We developed a novel ADC comprising an anti-CSPG4 antibody linked to a DNA minor groove-binding agent belonging to the novel pyrridinobenzodiazepine (PDD) class. Unlike conventional DNA-interactive pyrrolobenzodiazepine (PBD) dimer payloads that cross-link DNA, PDD-based payloads are mono-alkylating agents but have similar efficacy and substantially enhanced tolerability profiles compared to PBD-based cross-linkers. We investigated the anti-tumor activity and safety of the anti-CSPG4-(PDD) ADC in vitro and in human melanoma xenografts. Anti-CSPG4-(PDD) inhibited CSPG4-expressing melanoma cell growth and colony formation and triggered apoptosis in vitro at low nanomolar to picomolar concentrations without off-target Fab-mediated or Fc-mediated toxicity. Anti-CSPG4-(PDD) restricted xenograft growth in vivo at 2 mg/kg doses. One 5 mg/kg injection triggered tumor regression in the absence of overt toxic effects or of acquired residual tumor cell resistance. This anti-CSPG4-(PDD) can deliver a highly cytotoxic DNA mono-alkylating payload to CSPG4-expressing tumors at doses tolerated in vivo.

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Comparison in low-tumor-burden follicular lymphomas between an initial no-treatment policy, prednimustine, or interferon alfa: a randomized study from the Groupe d'Etude des Lymphomes Folliculaires. Groupe d'Etude des Lymphomes de l'Adulte.

Journal of Clinical Oncology ◽

10.1200/jco.1997.15.3.1110 ◽

1997 ◽

Vol 15 (3) ◽

pp. 1110-1117 ◽

Cited By ~ 346

Author(s):

P Brice ◽

Y Bastion ◽

E Lepage ◽

N Brousse ◽

C Haïoun ◽

...

Keyword(s):

Overall Survival ◽

Follicular Lymphoma ◽

Alkylating Agents ◽

Intensive Therapy ◽

Tumor Burden ◽

Interferon Alfa ◽

Response To Therapy ◽

Response To Treatment ◽

Survival Duration ◽

Overall Response

PURPOSE To evaluate prospectively in patients with follicular lymphoma and a low tumor burden three therapeutic options: delay of any treatment until clinically meaningful progression, immediate treatment with an oral alkylating agent, or treatment with a biologic response modifier, interferon alfa-2b. PATIENTS AND METHODS Newly diagnosed follicular lymphoma patients with a low tumor burden (n = 193) were randomly assigned to one of three arms: arm 1, no initial treatment (n = 66); arm 2, prednimustine 200 mg/m2/d for 5 days per month for 18 months (n = 64); or arm 3, interferon alfa 5 MU/d for 3 months then 5 MU three times per week for 15 months (n = 63). Clinical characteristics were similar in the three arms. RESULTS Overall response rates with prednimustine and interferon alfa were 78% and 70%, respectively. The overall response to therapy, when deferred, was similar at 70%. With a median follow-up duration of 45 months after randomization, the median freedom-from-treatment (FFT) interval was 24 months in arm 1 and the interval of freedom from treatment failure (FFTF) was 40 months in arm 2 and 35 months in arm 3. The median overall survival time was not reached and the overall survival rate at 5 years was 78% in arm 1, 70% in arm 2, and 84% in arm 3. Therefore, deferred treatment does not adversely influence survival at 5 years. Patients who progressed within 1 year had a significantly shorter survival duration (median, 48 months). CONCLUSION Delayed treatment is feasible in patients with follicular lymphoma and a low tumor burden. For patients with early progression, more intensive therapy should be considered. For others, because delay of treatment until significant clinical progression does not seem to hamper the prognosis or subsequent response to treatment, the long-term toxicity of alkylating agents can be reduced.

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Mood and clinical status in patients with multiple myeloma.

Journal of Clinical Oncology ◽

10.1200/jco.1991.9.12.2219 ◽

1991 ◽

Vol 9 (12) ◽

pp. 2219-2224 ◽

Cited By ~ 33

Author(s):

P M Silberfarb ◽

K M Anderson ◽

A C Rundle ◽

J C Holland ◽

M R Cooper ◽

...

Keyword(s):

Multiple Myeloma ◽

Regression Model ◽

Cox Regression ◽

Clinical Status ◽

Objective Response ◽

Disease Process ◽

Response To Treatment ◽

Survival Duration ◽

Data Set ◽

Physical Variables

Two hundred ninety patients with a recent diagnosis of multiple myeloma were studied psychologically at the time of initial treatment. Physician- and patient-completed psychosocial scales were correlated with physical variables used to measure tumor load and physical status. A logistic regression model was used to analyze objective response to treatment. Indirect measures of response to treatment were obtained, and factors influencing survival duration were studied using a Cox regression model. If physical variables were controlled, there were no significant correlations between psychologic scores on entry and response to treatment or survival duration. Thus, the notion that mood influences disease outcome once the disease process has begun in patients with multiple myeloma is not supported by this data set.

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MDR-1 expression and response to vincristine, doxorubicin, and dexamethasone chemotherapy in multiple myeloma refractory to alkylating agents.

Journal of Clinical Oncology ◽

10.1200/jco.1994.12.1.115 ◽

1994 ◽

Vol 12 (1) ◽

pp. 115-119 ◽

Cited By ~ 51

Author(s):

J J Cornelissen ◽

P Sonneveld ◽

M Schoester ◽

H G Raaijmakers ◽

H K Nieuwenhuis ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Plasma Cell ◽

Multiple Drug Resistance ◽

Labeling Index ◽

Cell Labeling ◽

Survival Duration ◽

Beta 2 ◽

Beta 2 Microglobulin ◽

Mdr 1

PURPOSE To assess whether the presence of enhanced multiple drug resistance (MDR)-1 gene expression in multiple myeloma (MM) patients predicts survival, as well as response to vincristine, doxorubicin, and dexamethasone (VAD) chemotherapy. PATIENTS AND METHODS Sixty-three MM patients refractory to alkylating therapy were studied. The presence of the MDR-1 gene product, a 170-kd glycoprotein (P-170), was analyzed in bone marrow plasma cells by means of the alkaline phosphatase (APAAP) technique using the P-170-specific monoclonal antibody (MoAb) C219. The prognostic value of MDR-1 gene expression, examined before VAD treatment, was compared with other established prognostic factors including beta 2-microglobulin, albumin, lactate dehydrogenase (LDH), and the plasma cell labeling index. RESULTS Fifty-nine percent of all samples were P-170-positive. No association could be demonstrated between response to VAD and MDR-1 gene expression (chi 2 P = .359), in contrast to high serum beta 2-microglobulin levels, which were positively correlated with response (P = .006). P-170-positive and -negative patients showed a median survival duration of 23 and 22 months, respectively, a difference that was not statistically significant (P = .9). beta 2-microglobulin, LDH, albumin, and the plasma cell labeling index were all significantly correlated with survival. CONCLUSION These results indicate that other mechanisms of resistance must be involved in MM apart from MDR. The role of MDR status at this stage of disease may be biased by the major contribution of dexamethasone to induction of response by VAD in MM patients.

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The mTOR Inhibitor RAD001 (everolimus) Is Active Against Multiple Myeloma Cells In Vitro and in Vivo.

Blood ◽

10.1182/blood.v104.11.1496.1496 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1496-1496 ◽

Cited By ~ 2

Author(s):

Nicholas Mitsiades ◽

Ciaran McMullan ◽

Vassiliki Poulaki ◽

Joseph Negri ◽

Noopur Raje ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Cells ◽

Cell Lines ◽

Stromal Cells ◽

Mtor Inhibitor ◽

Alkylating Agents ◽

Anti Tumor Activity

Abstract We have recently shown that tumor cell proliferation, survival and drug-resistance in multiple myeloma (MM) and a broad range of other tumors is critically influenced by insulin-like growth factors (IGFs) and their receptor (IGF-1R) (Cancer Cell2004;5:221–30). Among the pleiotropic signaling cascades downstream of IGF-1R activation, we focused on the functional implications and therapeutic targeting of the Akt/p70S6K/mTOR axis, particularly of mTOR (mammalian Target of Rapamycin), due to its regulatory role on cellular bioenergetics, a key aspect of tumor pathophysiology. Herein, we describe the in vitro and in vivo profiles of anti-tumor activity of the selective mTOR inhibitor RAD001 (Everolimus, Novartis AG). Using in vitro MTT assays, we observed that RAD001 is active (at nM concentrations) against a broad range of tumor cells, including >40 MM cell lines and >10 primary MM tumor cells (including cell lines or primary cells resistant to Dex, alkylating agents, anthracyclines, thalidomide (Thal), immunomodulatory Thal derivatives, bortezomib, and/or Apo2L/TRAIL), without significant impact on viability of normal hematopoietic cells or other normal tissues (e.g. bone marrow stromal cells), and its anti-MM effect was not blocked by forced overexpression of Bcl-2 or constitutively active Akt. While cytokine- or cell adhesion-mediated interactions with the bone marrow (BM) microenvironment (e.g. BM stromal cells) protects MM cells from conventional therapies (e.g. Dex or cytotoxic chemotherapy), RAD001 was able to overcome this protective effect in co-culture models of MM cells with BM stromal cells or in vitro MM cell exposure to survival factors, e.g. IL-6 or IGF-I. Furthermore, RAD001 sensitized MM cells to other anti-MM therapeutics, e.g. dexamethasone, cytotoxic chemotherapeutics, or the proteasome inhibitor bortezomib, even in cases of primary MM tumor cells refractory to these respective agents. Using hierarchical clustering analyses and relevance network algorithms, we found that the pattern of MM cell dose-response relationships to RAD001 is clearly distinct from the patterns of sensitivity or resistance to other conventional or investigational anti-MM drugs. This further supports the notion that RAD001 confers a constellation of pro-apoptotic/anti-proliferative molecular sequelae distinct from those of currently available anti-MM drugs, and also suggests that RAD001 may have anti-tumor activity even against subgroups of MM which may be resistant to other novel therapies which that are currently in clinical development. Importantly, administration of RAD001 in a SCID/NOD mice model of diffuse MM bone had in vivo anti-tumor activity, including suppression of MM tumor burden and prolongation of survival (p<0.01, log-rank test). These studies highlight an important role for mTOR in growth/survival of human MM cells and provide proof-of-principle for future clinical studies of mTOR inhibitors for the treatment of MM and other plasma cell dyscrasias.

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Bortezomib Targets Multiple Myeloma Endothelial Cells.

Blood ◽

10.1182/blood.v104.11.4903.4903 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4903-4903

Author(s):

Aldo M. Roccaro ◽

Teru Hideshima ◽

Noopur Raje ◽

Shaji Kumar ◽

Kenji Ishitsuka ◽

...

Keyword(s):

Multiple Myeloma ◽

Endothelial Cells ◽

Chorioallantoic Membrane ◽

High Dose ◽

Thymidine Uptake ◽

Similar Data ◽

Dose Dependent Fashion ◽

Dose Dependent

Abstract Introduction: Bone marrow (BM) angiogenesis is an important hallmark of multiple myeloma (MM) which correlates with progression. Although MM remains incurable despite conventional and high-dose chemotherapy, the proteasome inhibitor Bortezomib (Velcade, formerly PS-341), can overcome conventional drug resistance in vitro and in vivo and it has recently been FDA approved for treatment of relapsed and refractory multiple myeloma. Here we evaluated whether anti-angiogenesis may contribute to the anti-MM activity of PS-341. We examined the effect of PS-341 on the angiogenic phenotype of endothelial cells (ECs) isolated from BM of patients with MM. Methods: MMECs were extracted from BM of patients with active MM using a lectin-based method. The MMEC population contained >95% factor VIII-related antigen (FVIII-RA)+ and CD31+ cells, as assessed by fluorescence activated cell sorting (FACS). Contamination by macrophages and plasma cells was <5%, evaluated by FACS for CD14 and CD38 positivity, respectively, as well as by RT-PCR and Western blot for CD38. Viability, assessed by trypan blue was >90%. MTT assay and [3H] thymidine uptake were used to evaluate the effects of PS-341 on survival and proliferation, respectively, of MMECs. Proliferation of MM.1S cells cocoltured with MMECs was measured by [3H] thymidine uptake. Cytokine (IL-6, VEGF) levels were quantitated by ELISA. Other in vitro angiogenesis functions examined included chemotaxis, spreading on fibronectin (FN), and morphogenesis on Matrigel. Ongoing work is looking at the effect of PS-341 on angiogenesis in vivo by using a chick embryo chorioallantoic membrane (CAM) model. Results: PS-341, at concentrations achievable in the plasma of patients, inhibited in vitro MMEC and HUVEC functions related to angiogenesis, including proliferation, chemotaxis, spreading on FN, and capillary formation on Matrigel. All these functions were affected in a dose-dependent fashion. A significant concentration-dependent reduction of VEGF and IL-6 production was observed in the presence of PS-341, as demonstrated by ELISA. Importantly, binding of MM.1S cells to MMECs triggers tumor cell proliferation, and PS-341 inhibits proliferation of adherent MM.1S cells in a dose-dependent fashion. Similar data were demonstrated in HUVECs. Conclusions: These data therefore demonstrate that PS-341 acts both directly and indirectly against MMECs, another mechanism which may contribute to the anti-MM activity of PS-341.

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