Immunomodulatory Thalidomide Derivatives Research Articles

Integrated Molecular Landscape of Lesions in Cereblon and Its Pathway in Myeloma Patients: Insights into Mechanisms of Relapse from IMiD-Based Treatments

Mutations of Cereblon ( CRBN) or members of its pathway (CRBN-pathway genes, CRBNPGs) have been reported to exhibit increased frequency in multiple myeloma (MM) patients (pts) after relapse from regimens containing immunomodulatory thalidomide derivatives (IMiDs)±Dex. However, it is unclear if these mutations cause complete loss-of-function (LOF) of CRBNPGs in a way that can explain these relapses, given that, in preclinical studies, MM cells with only partial CRBN LOF retain substantial IMiD responsiveness. We therefore performed analyses integrating whole exome sequencing, RNA sequencing, copy number variation (CNV) and structural variant data of pts in the MMRF CoMMpass study who were treated with IMiD-based therapies. We included all pts with ≥2 paired bone marrow samples before and after an IMiD-based treatment (lenalidomide [LEN], thalidomide [T], pomalidomide [POM]) (baseline and relapse samples) and analyzed the patterns of molecular lesions of 42 CRBNPGs (based on Sievers et al. Blood 2019 and Jones et al. Leukemia 2021). To assess for alternative causes of relapse, a set of 42 known MM driver genes (defined in Ansari-Pour et al. Blood 2023) was analyzed in parallel. 177 pts of the IA22 CoMMpass dataset fulfilled inclusion criteria; 172, 89, and 15 pts received LEN-, POM-, or T-based treatment, respectively. 30 pts (16.9%) had at least one mutated CRBNPG at any timepoint (pts mut) and overall 24 CRBNPGs were mutated in at least one pt at any time. In 19 pts (63.3%), a CRBNPG mutation was present already at baseline; 11 pts (36.7%) developed a new CRBNPG mutation at relapse; and 2 pts (6.7%) had >1 CRBNPG mutated (2 and 5 genes, respectively). FAM83F (n=4; 12.5%) and IKZF3 (n=3; 9.4%) were the CRBNPGs most frequently affected. CRBN was mutated in 2 pts (6.7%) but without CNV loss, while full-length gene transcript was detected in both pts. In 4 cases, CRBN was proximal to chromosomal translocation breakpoints, but no mutations or absence of wild-type CRBN transcript were observed. In addition, no fusion transcripts involving a CRBNPG were detectable (at fusion fragment per million [FFPM]>5). Interestingly, when we examined an additional set of IA22 pts who did not undergo IMiD-treatment (n=14), the frequency of CRBNPG mutations observed (n=2, 14.3%; SALL4 and DDB1) was similar to the IMiD-treated pts. An increase in variant allele frequencies (VAF) of mutations for any CRBNPG occurred in 12 pts (37.5% of pts mut), however, VAF did not exceed 0.5. A preexisting mutation was no longer detectable at relapse in 8 pts (26.7%) and 3 pts showed a decline in VAF. Assessment of other known MM “driver” genes as possible explanation of relapse revealed mutations in 24 pts (80.0% of pts mut) at relapse: VAF of these genes showed an overall heterogeneous picture, with KRAS, BRAF, ARID1A, TP53 and IRF4 being among the most recurrently affected genes, consistent with their universal MM driver gene function. 10 of 11 pts (90.9%) who developed a new CRBNPG mutation and 10 of 12 pts (83.3%) with VAF increase showed partial response or better to IMiD treatment vs. 3 pts without clinical response (1 stable disease, 2 progressive disease). Only 2 of 19 (10.5%) pts with a CRBNPG mutation present at baseline were non-responsive to IMiD treatment. While 4 pts had a combined loss of one CRBNPG allele and mutation of another (for genes COPS8, COPS7B, DEPDC5, FAM83F), only 2 of these combined events occurred at relapse and with VAF values <0.4, suggesting that complete LOF of these genes was not present in a substantial fraction of the MM cell population at relapse. Most pts received combination treatment of IMiDs+proteasome inhibitor (PI), but we observed that preclinical CRISPR KO of CRBNPGs did not cause sensitization (or resistance) to PIs, suggesting that the genomic results for CRBNPGs in clinical samples were conceivably not skewed by PI use, e.g. via elimination of cells with complete LOF of CRBNPGs. Overall, in this integrated analysis, a minority of MM pts relapsing from IMiD-based treatments harbor genomic defects of CRBNPGs but these do not involve complete LOF for any such gene. Notably, new/enriched mutations in other genes, beyond CRBNPGs, with known roles as MM drivers cannot be excluded as alternative explanations for these relapses. Additional and more complex, genomic or non-genomic, mechanisms may account for relapse from IMiD-based regimens.

Role of carfilzomib in the treatment of multiple myeloma

The introduction of bortezomib, a first-generation proteasome inhibitor, changed the standard-of-care for newly diagnosed and relapsed multiple myeloma patients. The next generation of proteasome inhibitors, such as carfilzomib, provides a novel pharmacokinetic and pharmacodynamic profile. In vitro data suggest a more specific and irreversible inhibition of the proteasome. Based on the clinical trials conducted to date, carfilzomib has activity in heavily pretreated as well as bortezomib-refractory/relapsed patients. The safety profile, specifically a lower incidence of peripheral neuropathy, efficacy in the high-risk setting, as defined cytogenetically, and the durability of responses indicate a great potential for carfilzomib as a promising therapy. Several trials are underway involving carfilzomib in the newly diagnosed setting and in combination with other active myeloma drugs such as immunomodulatory derivatives of thalidomide, alkylating agents and targeted therapies such as histone deacetylase inhibitors. The introduction of this agent is yet another step in improving the overall outcome of multiple myeloma patients.

How to Overcome Myeloma Stem Cell Resistance to Therapy - Targeting the Stem Cell Niche

AbstractAbstract SCI-6The concept of cancer stem cells has attracted again intense research interest, as the drug resistance attributed to this infrequent subpopulation of tumor cells could explain how patients can relapse even after prolonged complete clinical, biochemical, radiologic or even molecular remissions. In multiple myeloma (MM), several aspects of the cancer stem cell concept remain to be elucidated, including the potential heterogeneity of this cell subpopulation or whether CD138+ expression is incompatible or not with a MM stem cell. As these questions are being resolved, emerging data highlight that the drug resistance of MM cells with clonogenic/stem cell-like features is heavily influenced by interactions with non-malignant accessory cells of the local microenvironment, including bone marrow stromal cells (BMSCs). Indeed, transcriptional signatures of “stemness”, as identified in normal stem cells or cancer stem cells from other neoplasias, are detected in the bulk population of MM cell lines and are upregulated after MM cell interaction with BMSCs. MM cell lines and primary tumor cells contain subpopulations with clonogenic potential, such as the side population (SP) cells. SP cells, detected by low intracellular accumulation of Hoechst 33342 dye (in contrast to the tumor's “main population” (MP), are considered an enriched source of tumor-initiating cells in diverse neoplasias and were detected among CD138− CD20+ CD27+ clonogenic cells in primary MM samples. Interaction with BMSCs increases the viability of SP cells and their percentage within the MM cell population. While interaction with BMSCs or other accessory cells of the microenvironmental niche suppresses the anti-MM activity of glucocorticoids, conventional chemotherapeutics and certain investigational agents, other agents (e.g. immunomodulatory thalidomide derivatives (IMIDs), such as lenalidomide) have increased activity against MM SP cells in the context of this tumor-microenvironment interaction. These observations suggest that MM cells with stem cell-like features exhibit functional plasticity depending on which specific microenvironmental niche they interact with. The Hedgehog, Wnt and Notch pathways, as well as regulators of chromatin remodeling, e.g. histone demethylases, have emerged as putative links between drug resistance, “cancer stemness” and how these functional outcomes are modulated by the local microenvironment in MM. These pathways, as well as embryonic stem cell-associated antigens (e.g. SOX2), represent intriguing targets for investigational therapies. However, clinical translation of such treatments has notable challenges, as conventional criteria for response assessment may not accurately reflect the treatment's impact on clonogenic tumor cells. Progression-free survival is considered a more appropriate endpoint for cancer stem-cell targeting agents, its assessment, however, may be confounded without concomitant treatment that suppresses the bulk of the tumor. Consequently, candidate cancer stem cell-targeting agents may have to be evaluated in combination with regimens (including lenalidomide-bortezomib-Dex (RVD) or other combinations built around the therapeutic “backbone” of proteasome inhibition and IMIDs) which potently target the bulk MM cell population and induce high rates of complete/near complete responses. Further improvements of this dual targeting of clonogenic and bulk tumor cells may be facilitated by recently developed high-throughput platforms (e.g. compartment-specific bioluminescence imaging, CS-BLI) which screen, in the presence vs. absence of stroma or other accessory cells, large numbers of anti-tumor agents and combinations thereof against the bulk tumor cell population or its clonogenic compartments. These new platforms will inform the rational design of regimens that will hopefully improve the long-term outcome of MM patients by suppressing a clonogenic/stem cell-like tumor compartment and circumventing microenvironment-dependent drug resistance. Disclosures:Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

Lenalidomide Enhances Clonogenic Activity, Proliferation and Erythroid Lineage Commitment of CD34+ Progenitor Cells While It Is Cytotoxic to CD34- Accessory Cells.

AbstractAbstract 1184Lenalidomide, a second generation immunomodulatory derivative of thalidomide, has demonstrated significant clinical benefit in myelodysplastic syndrome, multiple myeloma and B-cell malignancies. Its side effects include myelosuppression, particularly neutropenia, and impaired filgrastim induced stem cell mobilization in patients with myeloma. The precise mechanism of action of lenalidomide on hematopoietic and bone marrow microenvironment remains largely unknown. In this study, we investigated the effect of lenalidomide on survival, lineage commitment and differentiation of normal purified CD34+ hematopoietic stem cells.Mobilized peripheral blood progenitor cells (PBPCs) were obtained from a normal allogeneic stem cell donor with an informed consent, and were subjected to positive selection with the EasySep magnetic cell selection system to purify CD34+ cells (purity of 95%). Unsorted and CD34- accessory cells were used as controls.To assess the direct cytotoxic and proliferative effect of lenalidomide on purified CD34+ cells, lenalidomide (Celgene, NJ) was dissolved in DMSO and was diluted in culture medium to final concentration of 0.1, 1, 10, or 100 μM. Purified CD34+, CD34- and unsorted cells were plated in triplicates at a density of 2×104/mL in serum-free StemSpan® medium supplemented with growth factors and cytokines. 24 hours after plating the cells, lenalidomide or 0.1% DMSO as control was added. 72 hours following treatment, cells were counted with Beckman coulter counter and phenotypically analyzed using Accuri C6 flow-cytometer. The percentage of viable cells was determined using 7AAD staining. While no difference in viability was noted in the treated vs. untreated purified CD34+ cells, the viability of CD34- cells and unsorted cells was reduced to 30% and 40% respectively by lenalidomide. Contrary to our assumption, lenalidomide, in a dose dependant manner, induced proliferation of purified CD34+ cells. Maximal increase in proliferation was observed between 1mM and 10mM concentrations. The rate of growth plateaued beyond the 10mM concentration and 100mM concentration appeared to have no additional effect. At the peak of growth (10mM), the cell count nearly tripled. (Fig. 1) Lenalidomide appeared to be cytotoxic to CD34- and unsorted cells in a dose dependant manner. At 100mM concentration, the CD34- cell proliferation dropped by 4 fold and the unsorted cells by 7 fold.To examine the effect of lenalidomide on the clonogenic potential of purified CD34+ cells, 2,000 cells were seeded onto MethoCult® media (Stem cell technologies) supplemented with growth factors and cytokines. Cells were cultured in the presence of lenalidomide (0.1, 1, 10 and 100 μM) or 0.1 % DMSO as control. BFU-E, CFU-GM and CFU-mix were scored on day 14 with the use of an inverted microscope and standard morphological criteria. Significance of difference between groups was determined by student's t test for paired samples. Lenalidomide induced a significant increase in clonogenic activity of CD34+ cells (148 ± 27 colonies per 103 CD34+ cells, p = 0.01), most evident at the dose of 10 μM with enhanced formation of BFU-E (102 ± 23/ per 103 CD34+ cells, p = 0.01) but demonstrated no significant impact on CFU-GM (36 ± 14 per 103 CD34+ cells, p = 0.83) compared to control. (Fig. 2)This study demonstrates that lenalidomide has pronounced effects on both CD34+ and CD34- components of mobilized PBPCs. It enhances the proliferative and clonogenic potentials of CD34+ cells with lineage commitment towards erythroid differentiation. It also has cytotoxic activity towards CD34- cells. It is likely that neutropenia and impaired mobilization of stem cells in patients treated with lenalidomide is mediated via its impact on CD34- accessory cells in the bone marrow microenvironment. Further studies exploring the impact of lenalidomide on normal bone marrow stromal cells, T and NK cell mediated signaling are needed. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Recent advances of IMiDs in cancer therapy

Immunomodulatory derivatives of thalidomide (IMiDs) have been used for the treatment of myelodysplastic syndrome and multiple myeloma; however, the mechanism of action of IMiDs is largely unknown. The purpose of this review is to provide an overview of recent findings on the mechanism of action of IMiDs, its use as a new treatment modality for various hematologic malignancies, and problems associated with stem cell mobilization after lenalidomide treatment. Recent clinical trials revealed lenalidomide as a promising new agent for the treatment of follicular non-Hodgkin's lymphoma (NHL) and also diffuse large B-cell lymphoma. Pomalidomide was shown to be even more effective in refractory multiple myeloma than lenalidomide. New guidelines for the management of venous thromboembolism have been established. The chemokine receptor 4 (CXCR4) inhibitor, AMD-3100, is recommended for patients who have received lenalidomide and failed to mobilize stem cells after G-SCF and cyclophosphamide. Preclinical studies investigated the pleiotropic functions of IMiDs, with a particular focus on immune modulation, their effects on new targets, stem cells and disruption of plasma cell microenvironment interactions. More and more indications for the use of IMiDs in hematologic malignancies have been identified. In order to establish better clinical usage of IMiDs, it is of utmost importance to clarify the antitumor mechanism of IMiDs. Here, we provide a review on the recent advances in the development of IMiDs. New guidelines for venous thromboembolism prophylaxis and stem cell mobilization failure associated with lenalidomide treatment have been established.

Pomalidomide: A new IMiD with remarkable activity in both multiple myeloma and myelofibrosis

Pomalidomide: A new IMiD with remarkable activity in both multiple myeloma and myelofibrosis

A modular Phase I study of lenalidomide and paclitaxel in metastatic castration-resistant prostate cancer following prior taxane therapy

Lenalidomide, a highly potent immunomodulatory derivative of thalidomide, potentiates the action of paclitaxel in vitro against prostate cancer cell lines in co-culture with mononuclear cells. A modular Phase I study of lenalidomide and paclitaxel in men with metastatic castration-resistant prostate cancer (CRPC) was conducted to assess PSA kinetics with lead-in lenalidomide and the feasibility of the combination. Men with metastatic CRPC with prior taxane chemotherapy were planned for single-agent "lead-in" lenalidomide for 21/28 days at dose-levels: -1 (5 mg), 0 (10 mg), +1 (15 mg), +2 (20 mg), +3 (25 mg); followed by lenalidomide at the same dose and schedule in combination with weekly intravenous paclitaxel 100 mg/m(2) over 3 h on days 1, 8, 15 every 28 days utilizing a 3 + 3 dose-escalation design. Dose-limiting toxicity was observed in 4/6 patients with first-cycle combination therapy at the 10 mg dose-level and 3/6 patients at the 5 mg dose-level of lenalidomide, respectively. These included Grade 3 neutropenia precluding planned paclitaxel therapy (n = 3), grade 3 gastrointestinal toxicity (n = 2), chest pain (n = 1) and pulmonary embolism (n = 1). With lead-in lenalidomide, two patients with lymph-node dominant CRPC had a PSA-decline and regression in lymph node disease, respectively. Two of seven evaluable patients had PSA declines by 50% with combination therapy. Progression-free survival was 13 weeks (range 4-35 weeks). The high dose-limiting toxicity rates observed with lenalidomide and weekly paclitaxel require exploration of alternate dose-schedules of the combination in the second-line setting of CRPC. These early observations suggest distinctive toxicity and efficacy outcomes from thalidomide in combination with paclitaxel.

Targeting Cdc7 Kinase in Multiple Myeloma.

Abstract 3847Poster Board III-783 IntroductionMyeloma remains an incurable cancer and the development of new therapeutic approaches is of fundamental importance. A key feature of progressive myeloma is deregulation of the mechanisms that control progression through the cell cycle, in particular, the increase in the activity of S-phase promoting factors and the loss of cell cycle brakes such as the tumour suppressor protein p53. Moreover, high expression of Mcl-1, a potent anti-apoptotic Bcl2 family member, and defects in the p53 pathway are now known to be instrumental in inhibiting responsiveness to current and emerging myeloma therapeutics. The Cell division cyle 7 (Cdc7) kinase is an essential protein for the initiation of DNA replication and for cell cycle progression. Previous studies have indicated that Cdc7 inhibition can cause p53 independent apoptosis in several cancer models both in vitro and in vivo and currently Cdc7 inhibitors are entering clinical trials as experimental drugs. In this work we study the effects of the prototype Cdc7 protein kinase inhibitor, PHA-767491, in myeloma cells. In addition to targeting Cdc7, PHA-767491 also targets cyclin dependent kinase 9 (CDK9) and can downregulate Mcl-1 in a variety of cancer cell lines. Hypothesis: Given the molecular characteristics of progressive myeloma and the spectrum of activity of PHA-767491, we hypothesized that Cdc7 inhibition could be an effective treatment strategy in myeloma. Methods The Multiple Myeloma Research Consortium Validation-Core cell lines were treated with increasing doses of PHA-767491 as a single agent and in combination with drugs that currently constitute standards of care in myeloma treatment. These lines included lines resistant to conventional anti-myeloma agents (e.g. melphalan, dexamethasone, and doxorubicin) as well as novel anti-MM agents (e.g. immunomodulatory thalidomide derivatives). A number of lines were TP53 mutants. Similarly, CD138+ purified primary cells with clincal resistance to lenalidomide and bortezomib were treated with increasing doses of PHA-767491. Cell viability was measured by Cell TitreGlo 72 hours post treatment allowing IC50 calcuations. Combination indices were calculated as described by Chou et Talalay. Apoptosis was assessed by PI and Annexin V staining using flow cytometry and immunoblotting with relevant antibodies was performed for cell cycle and apoptotic proteins of interest. ResultsWe found that PHA-767491 blocks proliferation and induces apoptosis with an IC50 in the low micromolar range in six out of seven MMRC cell lines and in primary cells from patient (s) with clinical resistance to bortezomib and lenalidomide. The averaqe and median IC50 value of MMRC cell lines was 2.25μM and 2μM respectively. TP53 mutation did not alter sensitivity to PHA-767491. Following treatment of myeloma cells with PHA-767491 there was potent inhibition of phosphorylation of the replicative helicase Mcm2 at serine 40/41 (a specific biomarker of Cdc7 activity) and complete downregulation of Mcl-1 by 6 hours, with concomitant caspase 3 cleavage and apoptosis as assessed by accumulation of sub-G1 cells. Annexin V/PI staining confirmed these cells to be apoptotic. When used in combination, PHA-767491 showed synergistic activity with melphalan and doxorubicin (Combination Index IC50 – 0.3, 0.7 respectively), even in the highly doxorubicin-resistant(p170/MDR1 mediated) line RPMI-8226-Dox-40 (Combination IndexIC50 – 0.3). ConclusionWe suggest that Cdc7 inhibition with PHA-767491 or with small molecules that have similar properties could be an effective strategy in myeloma treatment and merits further evaluation. Disclosures:Montagnoli:Nerviano Medical Sciences Oncology: Employment.

Preclinical Studies in Support of Defibrotide for the Treatment of Multiple Myeloma and Other Neoplasias

Defibrotide, an orally bioavailable polydisperse oligonucleotide, has promising activity in hepatic veno-occlusive disease, a stem cell transplantation-related toxicity characterized by microangiopathy. The antithrombotic properties of defibrotide and its minimal hemorrhagic risk could serve for treatment of cancer-associated thrombotic complications. Given its cytoprotective effect on endothelium, we investigated whether defibrotide protects tumor cells from cytotoxic antitumor agents. Further, given its antiadhesive properties, we evaluated whether defibrotide modulates the protection conferred to multiple myeloma cells by bone marrow stromal cells. Defibrotide lacks significant single-agent in vitro cytotoxicity on multiple myeloma or solid tumor cells and does not attenuate their in vitro response to dexamethasone, bortezomib, immunomodulatory thalidomide derivatives, and conventional chemotherapeutics, including melphalan and cyclophosphamide. Importantly, defibrotide enhances in vivo chemosensitivity of multiple myeloma and mammary carcinoma xenografts in animal models. In cocultures of multiple myeloma cells with bone marrow stromal cells in vitro, defibrotide enhances the multiple myeloma cell sensitivity to melphalan and dexamethasone, and decreases multiple myeloma-bone marrow stromal cell adhesion and its sequelae, including nuclear factor-kappaB activation in multiple myeloma and bone marrow stromal cells, and associated cytokine production. Moreover, defibrotide inhibits expression and/or function of key mediators of multiple myeloma interaction with bone marrow stromal cell and endothelium, including heparanase, angiogenic cytokines, and adhesion molecules. Defibrotide's in vivo chemosensitizing properties and lack of direct in vitro activity against tumor cells suggest that it favorably modulates antitumor interactions between bone marrow stromal cells and endothelia in the tumor microenvironment. These data support clinical studies of defibrotide in combination with conventional and novel therapies to potentially improve patient outcome in multiple myeloma and other malignancies.

Immunomodulatory Derivatives of Thalidomide (IMiD)-Induced Neutropenia Is Associated with PU.1 Downregulation and Myeloid Maturation Arrest

Previous clinical trials have shown that immunomodulatory derivatives of thalidomde (IMiDs) including lenalidomide or pomalidomide induce high response rates in patients with relapsed or refractory multiple myeloma. However, the most dose limiting adverse effect of IMiDs is severe neutropenia. This is in contrast to our findings that IMiDs promote a shift in hematopoietic progenitor lineage commitment toward myeloid precursors with a subsequent maturation stop.In colony forming assays using human CD34+ hematopoietic precursors treated with IMiDs, we observed a highly significant increase in myeloid progenitor formation at the expense of erythroid colonies. Treatment of hematopoietic progenitors with IMiDs showed an upregulation of CD33, a marker for immature myeloid cells. Further, in vitro treatment with IMiDs resulted in downregulation of the transcription factor PU.1. PU.1 gene knock-down models show increased granulopoiesis with impaired neutrophil maturation, resulting in an accumulation of promyelocytes. In accordance with that, Cathepsin G as a promyelocytic marker was highly upregulated under treatment with IMiDs measured by high-density oligonucleotide microarray, quantitative RT-PCR, western blot and ELISA, confirming the maturation arrest of neutrophil granulocytes. To evaluate medullary changes in granulopoiesis in human subjects treated with lenalidomide, we evaluated sequential bone marrows from 6 patients comparing bone marrow features prior to treatment, during treatment, and at the time of grade 4 neutropenia. Marrows at the time of neutropenia showed an accumulation of myeloid precursors (myelocytes, metamyelocytes) supporting impaired myeloid maturation. In 5 out of 6 patients the myeloid:erythroid (M:E) ratio increased with an M:E median of 1.95 at pretreatment and 9.2 at the time of the neutrophil nadir. At the same time bone marrow cellularity was unchanged.In summary we show that IMiDs downregulate PU.1, a key transcription factor involved in granulocyte differentiation. The loss of PU.1 results in medullary accumulation of immature myeloid precursors, with corresponding neutropenia in the peripheral blood. Further high amounts of azurophilic granules of promyelocytes subsequently increase Cathepsin G and neutrophil elastase 2 resulting in enhanced platelet aggregation and a possible higher risk for venous thromboembolism. Our findings show that IMiDs do not induce neutropenia by marrow suppression but rather by transient block of maturation, which might be overcome by application of G-CSF.

A modular phase I study of lenalidomide and paclitaxel in metastatic castration-resistant prostate cancer with prior taxane therapy

13545 Background: Thalidomide may enhance the clinical activity of docetaxel and reverse taxane-resistance in prostate cancer. Lenalidomide, a highly-potent immunomodulatory derivative of thalidomide, potentiates the action of paclitaxel in-vitro against prostate cancer cell lines in co-culture with mononuclear cells. A modular Phase I study of lenalidomide and paclitaxel in men with metastatic castration- resistant prostate cancer (CRPC) was conducted to assess PSA-kinetics with lead-in therapy and the feasibility of the combination. Methods: Men with metastatic CRPC with prior taxane chemotherapy were planned for single-agent “lead-in” lenalidomide for 21/28 days at dose levels: -1 (5mg), 0 (10mg), +1 (15mg), +2 (20mg), +3 (25mg); followed by lenalidomide at the same dose/schedule in combination with weekly intravenous paclitaxel 100mg/m2 on Days 1, 8, 15 every 28 days. A 3+3 dose-escalation design was utilized to assess the maximum tolerated dose, defined as a dose-limiting toxicity (DLT) rate ≥ 0.33 during lead-in and one cycle of combination therapy. Enteric-coated aspirin 81mg and warfarin 2mg was recommended for thromboembolic prophylaxis. Results: See Table. Five patients had therapy discontinued for toxicity between 3–34 weeks Progression-free interval varied from 9–35 weeks (n=5). With lead-in lenalidomide, 1 of 9 evaluable patients had a minor PSA-decline; one patient with lymph-node dominant metastatic disease had a 40% decrease in bulky neck adenopathy followed by progression-free survival of 8 months with combination therapy. Conclusions: The high DLT rates observed with lenalidomide and paclitaxel require exploration of alternate dose-schedules of the combination in the second-line setting of CRPC. Cohort Patients Lenalidomide Paclitaxel DLT DLT [type/grade (G)] 1 3 0mg 100 mg/m2 1 - vomiting, diarrhea: G3 2 3 10mg 100 mg/m2 3 - neutropenia: G 3 - neutropenia: G 3 - chest pain: G 3 3 3 5mg 100 mg/m2 2 - infection/neutropenia: G 3 - dehydration/infection: G 3 4 1 5mg 80 mg/m2 * 1 - pulmonary embolism: G4 * A lower paclitaxel dose-level was studied after a protocol amendment. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Bayer, Onyx Bayer, Onyx Celgene

A review of lenalidomide in combination with dexamethasone for the treatment of multiple myeloma

Lenalidomide (also known as Revlimid((R)), CC-5013) is an immunomodulatory derivative of thalidomide and has more potent anti-tumor and anti-inflammatory effects than thalidomide. The molecular mechanisms of anti-tumor activity of lenalidomide have been extensively studied in multiple myeloma (MM) both preclinical models and in clinical trials. Lenalidomide: directly triggers growth arrest and/or apoptosis of drug resistant MM cells; inhibits binding of MM cells to bone marrow (BM) extracellular matrix proteins and stromal cells; modulates cytokine secretion and inhibits angiogenesis in the BM milieu; and augments host anti-tumor immunity. Lenalidomide achieved responses in patients with relapsed refractory MM. Moreover, lenalidomide with dexamethasone (Dex) demonstrates more potent anti-MM activities than Dex both in vitro and in randomized phase III clinical trials. Specifically, the combination improved overall and extent of response, as well as prolonged time to progression and overall survival, resulting in FDA approval of lenalidomide with Dex for therapy MM relapsing after prior therapy.

Cathepsin G, a Potent Platelet Aggregation Activator, Is Upregulated in Multiple Myeloma Patients during Treatment with Lenalidomide (Revlimid).

Lenalidomide induces impressive response rates in patients with multiple myeloma. Unfortunately, one of the major side effects of this therapy is the increased risk of thromboembolic events (TEE) up to 17%. The reason for a higher risk of TEE associated with Lenalidomide is not known, but it has been found that aspirin is very effective in preventing TEEs induced by thalidomide and lenalidomide (Rajkumar, 2005). Cathepsin G is a serine proteinase present in the azurophilic granules of polymorphonuclear neutrophils (PMNs) and is a strong platelet aggregation agonist with potency similar to thrombin. In the current study we investigated the effect of the immunomodulatory derivatives of thalidomide (IMiDs) including Actimid and lenalidomide on cathepsin G in vitro and in vivo. To investigate the effect of IMiDs in vitro CD34+ hematopoietic progenitors were cultured in the presence of IL-3, IL-6 and SCF supporting the development of granulocytes and treated with Actimid or DMSO (control). RNA and protein were extracted for the analysis of cathepsin G. A significant up-regulation of cathepsin G (7.7 fold) was detected in oligonucleotide gene array analysis after 3 days of treatment with Actimid as compared to control group. These results were confirmed by RT-PCR, which showed a 3.7 and 7.6 fold mRNA-increase on day 6 and day 10 of culture compared to control. Next we analyzed the cathepsin G levels of patients (n=10) treated with lenalidomide before treatment, on day 15 of each cycle (1–4) as well as 1, 2, and 3 month after stop of lenalidomide treatment. We observed a continuous significant increase of the mRNA levels of PMNs over the course of treatment (baseline: 1 fold, cycle 2: 1.7 fold, cycle 3: 4.8 fold, cycle 4: 20.7 fold). These data were confirmed by measuring cathepsin G levels in patient serum by ELISA. Cathepsin G significantly increased from a baseline mean of 53 ng/ml to 77.5 ng/ml (cycle 2), 129.2 ng/ml (cycle 3) and 145.5 ng/ml (cycle 4). After stop of the lenalidomide treatment we observed a time dependent normalization to baseline levels of the cathepsin G with 61 ng/mL (1 month post treatment), 42.5 ng/mL (2 months post treatment), and 49.2 ng/mL (3 months post treatment). Our data show that IMiDs up-regulate the potent platelet aggregation activator cathepsin G in hematopoietic cells and thereby might contribute to the development of TEE in patients receiving IMiD treatment. Based on our data TEE prophylaxis should be continued for at least 1 more month after stop of therapy. For the first time our results provide an explanation why aspirin is effective in preventing TEE in patients receiving IMiDs. Further studies are needed to determine whether cathepsin G helps to predict TEE. Inhibition of cathepsin G might be a potential therapeutic target for preventing the hypercoaguable state induced by IMiD treatment.

Anti-Myeloma Activity of the Dual Akt/p70S6K Inhibitor EXEL-6075.

Context: Akt plays a pivotal role in the survival and proliferation of multiple myeloma (MM) cells and functions as a central link between upstream signaling pathways, such as growth factor receptors (e.g. IL-6/IL-6R, IGF/IGF-1R) and the kinase PI-3K with their downstream signaling effectors, such as the multifunctional mTOR-p70S6K cascade. The pivotal role of Akt in these proliferative/anti-apoptotic cascades for MM has provided the impetus for development of small molecule inhibitors against the kinase activity of Akt. We show that the dual Akt/p70S6K kinase inhibitor EXEL-6075 has potent anti-MM activity in preclinical models.Methods/Results: We tested a panel of 15 human MM cell lines for in vitro response to EXEL-6075 using MTT colorimetric survival assays, which showed that the majority of MM cells responded to EXEL-6075 with IC50 values <0.5 μM, including a subset of 6/15 MM cell lines with IC50 values in the range of 0.1 μM. Importantly, <16 hrs of exposure to 0.5 μM of EXEL-6075 was sufficient to commit these EXEL-6075-sensitive MM cell lines to cell death. Peripheral blood mononuclear cells (PBMCs) remain insensitive to the drug up to 5 μM, consistent with a 1-log therapeutic window compared to the aforementioned sensitive MM cell lines. Other non-malignant tissue, such as bone marrow stromal cells (BMSCs) and immortalized human hepatocyte cells were even less sensitive to EXEL-6075 (IC50>5 μM), further underscoring its selectivity towards neoplastic cells. EXEL-6075-responsive cells included MM cells resistant to diverse conventional and novel agents, such as dexamethasone, bortezomib, and immunomodulatory thalidomide derivatives, suggesting that dual Akt/p70S6K inhibition may overcome mechanisms of constitutive resistance to other currently available therapeutics. EXEL-6075 was also able to overcome the protective effects of IGF-1 and IL-6 on MM.1S cells. In addition, there was modest, if any, protection conferred to MM cells by BMSCs at low drug doses. Interestingly, when we compared the in vitro activity of EXEL-6075 against cell lines from MM vs. other neoplasias (including 11 epithelial cancer cell lines which also exhibited dose-dependent response to this kinase inhibitor), EXEL-6075 was significantly more active against MM cells, as evidenced by the distribution of IC50 values and AUCs of the dose-response curves for each group of cell lines (p=0.028, t-test). Ongoing studies are utilizing gene expression profiles of MM cells highly- vs. moderately-responsive to EXEL-6075, in order to identify putative markers of sensitivity vs. resistance to this kinase inhibitorConclusion: Targeting the Akt-mTOR-p70S6K pathway is an attractive strategy for the treatment of MM due to the central role Akt plays in proliferation and survival of MM. Inhibition of Akt using the small molecule inhibitor EXEL-6075 resulted in sub-μM killing of MM cell lines with > 1-log differential activity against the non-neoplastic tissues tested in our study. This remains an interesting strategy for the treatment of MM. Future in vivo studies will be needed to confirm these interesting in vitro results.

Activity of New Heat Shock Protein 90 (hsp90) Inhibitor NVP-AUY922 Against Myeloma Cells Sensitive and Resistant to Conventional Agents.

Context: The molecular chaperone hsp90 is a major anti-cancer therapeutic target because it regulates the function of proteins with pivotal roles in tumor cell proliferation, survival and drug resistance, including mutated/chimeric oncoproteins or oncogenic kinases/receptors. Our preclinical studies on the ansamycin hsp90 inhibitor tanespimycin (17-AAG) provided the rationale for clinical trials, either alone or in combination with the proteasome inhibitor bortezomib, for treatment of relapsed/refractory MM. In this study, we report preclinical studies of the new, non-ansamycin, hsp90 inhibitor NVP-AUY922.Methods/Results: We tested 36 human MM cell lines and observed with MTT colorimetric survival assays potent time- and dose-dependent anti-MM activity of NVP-AUY922. IC50 values were <12.5 nM for 31 of 35 cell lines, which were also more sensitive than all non-malignant cells tested with NVP-AUY922, including bone marrow stromal cells (BMSCs), immunortalized human hepatocytes and normal donor PBMCs, indicating a differential selectivity of NVP-AUY922 against neoplastic tissues vs. non-malignant cells. Importantly, MM cell lines or primary MM tumor cells resistant to dexamethasone (Dex), melphalan, immunomodulatory thalidomide derivatives (IMIDs), bortezomib or TRAIL were sensitive to NVP-AUY922. In addition, MM-1S cells constitutively over-expressing Akt were equally sensitive to NVP-AUY922 compared to their parental cell line. These anti-MM effects were rapid, as MM cells were committed to cell death within 16 hrs of exposure to only 25 nM of NVP-AUY922. NVP-AUY922 overcame the protective effects conferred to MM cells by exogenous IL-6 and IGF-1, while BMSC co-culture attenuated NVP-AUY922 activity only at doses <20 nM, as evidenced by compartment-specific bioluminescence imaging (CS-BLI). Mechanistic studies of NVP-AUY922 treatment of MM-1S cells showed early G2/M arrest followed by increased cell death, accompanied by caspase-3 and -8 cleavage (as early as 16 hrs). These events were preceded by decreased levels of Akt, B-Raf, phospho-MEK, cIAP2, and XIAP; and compensatory upregulation of hsp27 and hsp70. Given the highly single-agent activity of NVP-AUY922 at even low nM concentrations, formal statistical documentation of synergy was not observed in NVP-AUY922 combinations with conventional (e.g. Dex, doxorubicin) or novel (e.g. bortezomib) anti-MM agents. Encouragingly, no evidence of antagonism with any of these combinations was observed, indicating that NVP-AUY922 can be combined with current anti-MM agents in clinical settings.Conclusion: The new hsp90 inhibitor NVP-AUY922 has potent in vitro activity against MM cells resistant to conventional therapeutics, with selectivity for malignant compared to normal cells. Ongoing in vivo experiments and studies to identify biomarkers of pronounced sensitivity to NVP-AUY922 will help provide a framework for potential clinical trials of NVP-AUY922 in MM and other neoplasias.

Adverse Effects on Hemostatic Function of Drugs Used in Hematologic Malignancies

The adverse effects of drugs used in the treatment of hematologic malignancies are among the many factors contributing to the increased risk of both thrombosis and bleeding. These effects most often occur when combination of drugs are given. Some, such as L-asparaginase, result in both bleeding and thrombosis. Consideration must be given also to the bleeding or prothrombotic risk of the underlying hematologic disorder. The commonly used drugs with adverse effects on hemostasis include L-asparaginase, corticosteroids, inhibitors of vascular endothelial growth factor, gemtuzumab ozogamicin, thalidomide, and immunomodulatory derivatives of Thalidomide, and the hematopoietic growth factors. In addition, the syndrome of thrombotic microangiopathy may be brought on by several other drugs. Thus, a full understanding of these adverse effects is necessary in treating these disorders.

Lenalidomide and Bortezomib Inhibit Osteoclast Differentiation and Activation in Multiple Myeloma: Clinical Implications.

Osteolytic bone disease in Multiple Myeloma (MM) is caused by enhanced osteoclast (OCL) activation and inhibition of osteoblast function. The proteasome inhibitor bortezomib (PS341, Velcade) has potent anti-myeloma activity with impressive clinical responses. A recent study indicated that bortezomib has inhibitory effects on OCL (ASH 2005, Abstract #2488). Lenalidomide (CC-5013, Revlimid) is an immunomodulatory derivative of thalidomide that has shown promising anti-MM effects in patients with relapsed or refractory MM (Richardson et. al, Blood Jul 06). Significantly, a phase I clinical trial showed that lenalidomide and bortezomib could achieve responses in the majority of patients with MM, refractory to either agent alone (ASH 2005, Abstract #365). However, the effect of lenalidomide on human OCL lineage is unknown. Here we investigated the effect of lenalidomide and bortezomib on human OCL. Peripheral blood mononuclear cells (PBMC) from MM patients (n=11) and healthy donors (n=5) were stimulated with receptor activator of NFk-B ligand (RANKL) (50ng/ml) and M-CSF (25ng/ml) for two weeks to induce OCL formation, in the presence or absence of lenalidomide or bortezomib. OCL were identified by flow cytometric analysis using anti-aVb3 integrin. Lenalidomide and bortezomib inhibited OCL differentiation in a dose-dependent manner (n=13, median control: 70.9% at 0 μM; 63% at lenalidomide 2μM and 45% at 10μM; 35% at bortezomib 2nM and 11% at 5nM). TRAP staining (tartrate-resistant acid phosphatase) was performed to identify OCL and confirm OCL activity. Lenalidomide, as well as bortezomib inhibited OCL in a dose-dependent manner, as evidenced by a marked decrease in TRAP+ cells. To assess bone resorption activity, OCL were cultured with dentine discs, in the presence or absence of lenalidomide and bortezomib, followed by light microscopic analysis and additional measurement of soluble collagen I fragments from the supernatant. Both lenalidomide and bortezomib inhibited bone resorption in a dose-dependent manner. We next asked whether mature OCL were affected. OCL were induced by cytokine stimulation for 3 weeks and treated for 72h, followed by flow cytometry. Neither lenalidomide nor bortezomib altered total number of aVb3 integrin-expressing mature OCL (n=6). In addition, OCL culture supernatants were collected, and two major MM growth and survival factors produced by OCL, B-cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL), were measured by specific ELISA. Both lenalidomide and bortezomib strongly inhibited secretion of BAFF and APRIL. Finally, we determined whether lenalidomide and bortezomib inhibited expression of transcriptional factors important for OCL differentiation and survival. Cell lysates of CD14-expressing monocytic cells from normal donor PBMCs were subject to immunoblotting. Importantly, lenalidomide inhibited OCL differentiation by downregulation of PU.1 expression. These results therefore indicate, that lenalidomide and bortezomib inhibit OCL differentiation, thereby directly preventing the development of new osteolytic lesions. Moreover, BAFF and APRIL secretion by OCL is downregulated, thereby inhibiting MM cell survival in the bone marrow microenvironment.

Anti-Myeloma Activity of the Small-Molecule Aurora Kinase Inhibitor VE465.

Multiple Myeloma (MM) remains an incurable plasma cell neoplasia, despite recent additions in the therapeutic arsenal for its management. Aurora kinases play integral roles in the orchestration of chromosomes and cytoskeletal mobility during the process of cell division. Aurora kinase activity has been implicated in several tumor types, including ovarian, colon, and prostate cancers. To determine whether inhibition of Aurora kinase activity could attenuate myeloma cell survival, we performed studies of the Aurora kinase inhibitor VE465 (Vertex Pharmaceuticals / Merck & Co., Inc.). VE465 inhibits all 3 Aurora isoforms (Aur A, B and C) with approximate Ki values of 1, 26, and 8.7 nM respectively. MTT colormetric survival assays (72–96hrs exposure) showed that VE465 is active against a wide panel of human MM cell lines: 26 of 38 MM cell lines had IC50 values at or < 100 nM, which are significantly lower than IC50 values for normal hematopoietic cells, e.g. unstimulated or PHA-stimatuled PBMCs. Importantly, VE465 was active in vitro against MM cell lines and/or primary MM tumor cells resistant to various anti-MM therapeutics, including dexamethasone, alkylating agents, anthracyclines, the proteasome inhibitor bortezomib, and/or immunomodulatory thalidomide derivatives (IMiDs). Moreover, VE465 maintained its activity despite the presence of protective bone marrow-derived cytokines (e.g. IL-6). PI cell cycle analyses showed that VE465 causes (even within 8 hrs of treatment) caused pronounced G2 arrest, followed by significant shift of MM cells to sub-G1 gate, consistent with cell death. Immunoblotting analyses confirmed that VE465 treatment induces cleavage of PARP, as well as cleavage of caspases-8 and -9, without significant changes in the expression levels of several key molecular effectors (e.g. Mcl-1, Bax, p53, hsp70, hsp90, hs27) which have been previously implicated in the mechanism of anti-MM activity of diverse other therapeutics. Screening of VE465-based combination regimens with other anti-MM agents showed additive effects of VE465 with the histone deacetylase inhibitor Vorinostat (SAHA) (Merck & Co., Inc). Ongoing studies in our Center are addressing the identification of specific molecular markers correlating with the degree of sensitivity of MM cells to VE465. Our in vitro evidence for induction of MM cell death and therapeutic window for the anti-MM effect of VE465, its ability to overcome protective effect of BM-derived cytokines, and the clearly distinct pattern of molecular sequelae of VE465 compared to several other agents in our current anti-MM therapeutic armamentarium, all suggest that Aurora kinase inhibition represents an intriguing novel targeted treatment strategy in MM. Importantly, these studies, particularly the identification of a sizeable subset of MM cell lines with higher sensitivity to VE465 than normal cells, provide the framework for in vivo VE465 studies in progress, alone and in combination with other anti-MM agents, to inform the design of potential clinical trials of this class of agents for MM.

Reversine: A Chemical Probe Providing Insight into Myeloma Biology and Potential Therapeutic Applications.

Multiple myeloma (MM) is proposed to be driven by a CD138− CD20+ population of “MM stem cells” (MMSCs) which give rise to the more “differentiated” CD138+ malignant plasma cells (PCs), which have historically been the main target of anti-MM therapeutics. We hypothesized that inhibition of “differentiation” of these MMSCs to CD138+ derivative MM PCs could allow for expansion of the MMSC compartment and facilitate its cellular and molecular characterization. To test this hypothesis, we performed in vitro studies of MM cells (cell lines and primary MM cells) with reversine (2-(4-Morpholinoanilino)-6-cyclohexylaminopurine), a small molecule known to suppress differentiation (e.g. of myoblasts to myocytes) and/or cause de-differentiation of lineage-committed cells. We observed no reversine-induced expansion in CD138− populations deemed to represent MMSCs and no de-differentiation of CD138+ CD20− MM PCs to more “immature” CD138− CD20+ phenotype. Instead, sub-uM doses of reversine (as low as 0.1 uM, and significantly lower than the >5 uM required for its de-differentiation effects in other tissue type models) triggered major increases in MM cell death, including cells resistant to conventional (e.g. Dex, alkylating agents, anthracyclines, thalidomide, bortezomib, immunomodulatory thalidomide derivatives) and/or investigational agents (e.g. Apo2L/TRAIL) anti-MM agents. PBMCs, stromal cells, and immortalized hepatocytes were less sensitive to reversine than MM cells. Reversine was also active against a broad panel of neoplastic cells, e.g. various leukemia and lymphoma cell lines, suggesting a potentially broader spectrum of anti-neoplastic properties. Reversine was active against MM cells which are independent from or unresponsive to bone marrow stromal cell (BMSC) and was also able to overcome the effect of cytokine- (e.g.IL-6, IGF-1) or stroma-derived protection to BMSCs-responsive MM cells. The anti-MM effect of reversine involved G2 cell cycle arrest and commitment to cell death by drug exposure as short as 8hrs. Cleavage of PARP and caspase 3 were observed, and protein levels for diverse signaling effectors in the B-Raf and Akt cascades were decreased. Reversine enhanced responses of primary MM cells to other anti-MM agents, e.g. Dex, and had additive effects with cytotoxic chemotherapeutics or bortezomib. Reversine affected the molecular profiles of MM cells in a manner distinct from other anti-MM agents studied to date. Unlike bortezomib, reversine did not decrease NF-κB transcriptional activity, or affect the levels of several Bcl-2 family members, inhibitors of apoptosis, or heat shock proteins. These data suggest that reversine kills MM cells in an NF-κB independent manner, and also supports a broader conclusion that reversine has distinct profiles of molecular effects and anti-MM activity. Differential transcriptional profiling of MM cells with >1 vs. <1 log differential sensitivity to the normal tissues tested identified candidate markers correlating with increased reversine sensitivity, including molecules with known roles in malignant transformation, e.g. PKCiota, polo-like kinase-1 and c-myb. These results, coupled with ongoing in vivo studies, suggest that reversine may represent a distinct class of agents with therapeutic potential for specific subset(s) of MM and perhaps other hematologic neoplasias.

Phase I trial of three-weekly Docetaxel, Carboplatin and oral lenalidomide (Revlimid®) in patients with advanced solid tumors

Lenalidomide is an immunomodulatory derivative of thalidomide with significantly greater in vitro activity and a different toxicity profile. In preclinical trials it has shown synergy with chemotherapy. Primary objective of this study was to determine the maximum tolerated doses of docetaxel and carboplatin when combined with oral lenalidomide in a standard phase I study design. Between September 2004 and May 2005, 14 patients with pathologically proven solid tumors, < or =2 prior chemotherapy regimens, performance status ECOG 0/1, and adequate organ function were enrolled. Dose limiting toxicities (DLT) were defined as > or = grade 3 non-hematological, or grade 4 hematological toxicity. No growth factors were used during cycle 1. Three of four patients treated at dose level 1, docetaxel 60 mg/m(2) and carboplatin AUC 6 on Day 1, and lenalidomide 10 mg orally daily on Days 1-14 of a 21 day cycle experienced DLT (grade 3 electrolyte changes in two patients, and grade 4 neutropenia in one patient). Ten patients were treated at dose level -1, docetaxel 60 mg/m(2) and carboplatin AUC 6 on Day 1, and lenalidomide 5 mg orally daily on Days 1-14 of a 21 day cycle with one DLT (Grade 4 neutropenia). There were no treatment-related deaths or irreversible toxicities. Of the 14 response-evaluable patients, five achieved a partial response (5 out of 9 patients with non-small cell lung cancer. Docetaxel 60 mg/m(2) and carboplatin AUC 6 on Day 1, with lenalidomide 5 mg orally daily on Days 1-14 days of a 21 day cycle is the maximum tolerated dose without the use of prophylactic growth factors. This combination is active and further evaluation in a phase II trial is warranted.