Antiproliferative Effect Of Lenalidomide Research Articles

Can Monosomy 7 be Targeted By Next Generation Cereblon-Modulating Agents?

Genomic advances have initiated a new era of precision oncology. Targeted therapeutics can rationally select potentially responsive patient populations and exclude resistant patients based on molecular mechanisms of action. The application of immunomodulatory drugs (IMiDs) evolved from unselective usage to the identification of del(5q) as a target, which then led to mechanistic clues and clarification of its mode of action. Lenalidomide (LEN) has been used in non del(5q) patients with a variable response rate, but the molecular underpinnings for LEN efficacy in this setting are not known. Genomic analyses (Negoro et al., Leukemia 2016) have provided only a marginal improvement in response prediction, suggesting that other factors might be relevant in sensitizing cells to LEN. The effects of LEN are related to its binding affinity to cereblon (CRBN), the substrate adaptor of the CRL4CRBN E3 ubiquitin ligase, a cullin-ring ligase consisting of damaged DNA-binding protein1(DDB1), cullin4a (CUL4A), and regulator of cullins-1 (ROC1). LEN selectively activates CRL4CRBN E3 ubiquitin ligase to selectively degrade CSNK1A1 leading to synthetic lethality in patients with del(5q) that are haploinsufficient (HI) for CSNK1A1. However, LEN and the new generation compounds pomalidomide and iberomide (CC-220) also trigger recruitment and degradation of the zinc finger transcription factors IKAROS (IKZF1, 7p12.2) and AIOLOS (IKZF3, 17p12-q21.1) through CRBN, and therefore promote their proteosomal degradation. The synthetic lethality of these immunomodulator agents relies on expression levels of AIOLOS and IKAROS. Response to those agents depends strictly on low expressions of AIOLOS and IKAROS sensitizing certain cell types (e.g., myeloma). We hypothesized that levels of LEN sensitivity might correlate with expression levels of its targets in selected disease subtypes of myeloid neoplasia (MN). We studied a large cohort of patients with MN (n=3,328) and we identified 122 patients with -7 and 194 patients with del(7q). We noted that for those cases with informative RNA-seq expression, IKAROS exhibited haploinsufficient (HI) mRNA levels in 67% of patients with -7 and 40% in del(7q), as defined based on levels <25%tile of the diploid expression of cases (Kerr, ASH 2019). Since IKAROS is described to be a target of iberomide, we hypothesized that monosomy 7 (-7) but not del(7q) could selectively benefit from this agent, analogous to LEN in del(5q). Consistent with this proposition we further selected 160 hematopoietic cancer cells with available IKZF1 CNV status in a publicly available database of cell drug sensitivity (cancerrx). For all cell lines, the anti-proliferative effect of LEN was described. Cells with monosomy of IKZF1 (CNV=1) had an increased sensitivity to LEN as their IC50 was lower than compared to that of cells with IKZF1 with CNV ≥ 2 (6.1μM vs. 24.3μM). To further validate this prediction, we revisited the responsiveness to LEN (defined by Negoro et al., Leukemia 2016) of non-del(5q) MDS patients (n=79) showing an ORR of 40%. By focusing on patients with alterations in chr7 we identified 10 patients with MDS and high or very high International Prognosis Scoring System-Revised (IPSS-R) risk [6 patients with -7 and 4 patients with del(7q)]. In this cohort LEN response was assessed at 3 and 6 months; 4 out 6 patients with -7 responded to LEN, while del(7q) patients were all refractory (n=4). Responding monosomy 7 patients with complete bone marrow response (n=3) received 10 or 50mg doses of LEN (2 patients had a complex karyotype and 1 had isolated -7). The remaining responding patient had a partial response with hematological improvement. Interestingly, this patients was treated with a 5mg dose of LEN and had a TP53 mutation. In the other 2 patients: one with -7, LEN was discontinued because of pancytopenia associated with treatment, while in the other one -7 could not be confirmed by FISH. In the group of del(7q) patients, complex karyotypes and the presence of del(5q) in 2 did not predict for better response. Our preliminary results suggest that selective application of new generation IMiDS targeting IKAROS have clinical potential in patients with -7. Supportive data from in vitro cellular models will be comprehensively presented. Disclosures Walter: MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nazha:Tolero, Karyopharma: Honoraria; Incyte: Speakers Bureau; Abbvie: Consultancy; Jazz Pharmacutical: Research Funding; Daiichi Sankyo: Consultancy; Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

Lenalidomide In Combination with the Activin Receptor Type II Murine Fc Protein RAP-011: Preclinical Rationale for a Novel Anti-Myeloma Strategy

AbstractAbstract 4075The introduction of novel treatment strategies targeting tumor cells within their microenvironment have resulted in prolonged survival for Multiple Myeloma (MM) patients. Lenalidomide belongs to this category of agents working via tumoricidal, anti-osteoclast and immunomodulatory activities. However, lenalidomide lacks bone anabolic effects. We have recently reported that activin A mediates osteoblast inhibition in MM and neutralizing activin A via a soluble receptor, RAP-011 (murinized form of ACE-011) (Acceleron Pharma, Cambridge, MA), restores bone architecture and reduces tumor burden in vivo. We therefore hypothesized that the combination with RAP-011 may potentiate lenalidomide effects and vice versa as they act via complementary mechanisms.Our previous data demonstrates that 50% of MM patients have increased bone marrow (BM) levels of activin A that correlate with osteolytic burden. Here, we observed that 2 and 10 μ M lenalidomide (Selleck Chemicals, Houston, TX) upregulated activin A in 3 out 6 bone marrow stromal cell (BMSC) samples by 1.9 and 2.8 fold (average ± st.dev. 1052 ± 190 and 1667 ± 732 pg/ml respectively, compared to 734 ± 553 in control, p=0.2). There was no time-dependent upregulation of activin A. Of note, no augmentation of activin A was noted in BMSC which already expressed high baseline levels of the cytokine (average ± st.dev 3638 ± 3755 pg/ml in control vs 3074 ± 2997 after lenalidomide 10 μ M).Previous data suggest that high concentrations of activin A induce growth arrest and apoptosis in myeloma and breast cancer cells and may therefore mediate lenalidomide cytotoxicity. To ensure that inhibition of activin would not antagonize lenalidomide anti-tumor effects, we investigated whether activin A inhibition affected the cytotoxic and anti-proliferative effects of lenalidomide on MM cells alone and in co-culture with BMSC. As previously demonstrated, RAP-011 did not exert any direct anti-tumor effects. Lenalidomide 10 μ M induced between 20 and 40% of apoptosis in several myeloma cell lines, such as MM1.S, LR5, DOX40 and RPMI, independent of activin A inhibition. Similarly, lenalidomide almost completely reversed the proliferative advantage conferred by BMSC to tumor cells. Combining lenalidomide and RAP-011 was not antagonistic to the inhibition of the proliferative advantage conferred by BMSC to myeloma cells, suggesting that lenalidomide's direct anti-tumor activity is not mediated through activin A.Finally we assessed OB differentiation in the presence of both RAP-011 and lenalidomide. We have previously reported that activin A inhibitory effects on OB differentiation are reversed by RAP-011 treatment. Here, we noted diminished alkaline phosphatase (ALP, a marker of osteoblast activity) expression during osteoblastogenesis in the presence of increasing concentrations of lenalidomide (17% ± 3 decrease by 2 μ M and 26% ± 11 by 10 μ M compared to control, p=0.01 and 0.06 respectively). In contrast, combination with RAP-011 restored the osteogenic potential by increasing ALP expression close to control levels in healthy donor-derived BMSC and above control levels in MM-derived BMSC.These preliminary data suggest that lenalidomide results in upregulation of activin A expression in MM BMSCs which have low baseline levels. Combining lenalidomide with RAP-011 results in restored osteogenesis presumably by inhibiting activin signaling. Importantly, we observed no antagonistic effect of RAP-011 on lenalidomide's anti-tumor activity, confirming that activin A does not mediate the anti-tumor activity of lenalidomide. Ongoing in vivo studies using a murine model of myeloma will confirm the efficacy of this promising combination. Our results provide a preclinical rationale for combining lenalidomide with ACE-011 to target myeloma by manipulating the microenvironmental compartment, specifically the bone compartment. Disclosures:Seehra:Acceleron Pharma: Employment. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Raje:Celgene, Novartis: Consultancy; Astrazeneca, Acetylon: Research Funding; Celgene, Amgen: Membership on an entity's Board of Directors or advisory committees.

Lenalidomide Inhibits Multiple Myeloma Cell Proliferation in Vitro Via Its Effect On Expression of Oncogenes and Tumor Suppressor Genes.

Abstract 2855Poster Board II-831Lenalidomide is an oral anti-proliferative and immunomodulatory drug. In combination with dexamethasone, it is indicated for the treatment of patients with multiple myeloma (MM) who have received at least one prior therapy. In this study we investigated direct antimyeloma effects of lenalidomide in vitro using a panel of human MM cell lines with various cytogenetic features and bone marrow cells from patients with active MM. We also assessed the effect of lenalidomide on expression of tumor suppressor and enhancer genes such as p21cip1, SPARC, ING1/4, p57kip2, p53, cyclin D1/2, IRF4/MUM1 and IRF8/ICSBP. At attainable plasma levels in treated patients, lenalidomide directly inhibited human MM cell proliferation. Lenalidomide strongly increased expression of tumor suppressor genes such as p21waf1/cip1, SPARC, IRF8, ING4 and p57kip2. In the MM cell lines tested, lenalidomide had partial but consistent inhibitory effects on expression of IRF4, an important MM survival factor. However, lenalidomide had no marked effect on expression of tumor enhancer genes such as VEGF, cyclin D1/2 and MAF or tumor suppressor genes such as ING1 and p53 in most lines of cells. This suggests that the antiproliferative effects of lenalidomide on MM cells may be related to the upregulation of some tumor suppressor gene expression. Statistical analyses show that the antiproliferative effect of lenalidomide is significantly correlated with the drug induced upregulation of SPARC and IRF8 expression (p=0.0016; p=0.012, respectively), but not with the drug induced changes of p21 (p> 0.05) and IRF4 expression (p> 0.05). Furthermore, the antiproliferative effect of lenalidomide was significantly correlated with the constitutive expression levels of cyclin D1 (p=0.021) and IRF4 (p=0.027), and inversely correlated with the constitutive level of cyclin D2 (p=0.041) in these MM cell lines. Using bone marrow myeloma cells from patients, we confirmed that the sensitivity of cells to lenalidomide was associated with SPARC and IRF8 upregulation and baseline levels of cyclin D1/2 and IRF4 expression. Using MM cell lines adapted to prolonged exposure (5 months) to lenalidomide, we found that cells became resistant to the drug in association with decreased baseline levels of cyclin D1 and IRF4. In conclusion, lenalidomide demonstrates direct inhibitory effects on proliferation various MM cells. These antimyeloma activities may help explain the clinical efficacy seen in patients treated with lenalidomide. Lenalidomide treatment of MM cells increased SPARC and IRF8 mRNA expression, whereas pre-treatment cyclin D1/2 and IRF4 mRNA levels were associated with increased sensitivity and may have prognostic potential for MM therapy with lenalidomide. Disclosures:Zhang:Celgene Corporation: Employment. Adams:Celgene Corporation: Employment. Kosek:Celgene Corporation: Employment. Schafer:Celgene Corporation: Employment. Bartlett:Celgene Corporation: Employment.

The Anti-Proliferative Effect of Lenalidomide On MM Cells in Vitro Is Ameliorated by Prior Exposure to Pomalidomide, An Agent with Activity against Lenalidomide Resistant MM Cells.

Abstract 4926 IntroductionDespite recent advances in the treatment of Multiple Myeloma (MM) the disease remains incurable and initial response to therapy is followed inexorably by relapse, chemoresistance and death. Therefore, relapsed and refractory disease remains an area of high unmet need with an urgent need for new therapies that are able to exert significant effects on the tumor. Lenalidomide (Len), an oral anti-proliferative and immunomodulatory drug, has been approved in combination with dexamethasone (Dex) for previously treated MM. Early clinical results also suggest that pomalidomide (Pom) is effective in the treatment of relapsed and refractory MM, including patients who have previously been treated with Len. The achievable plasma levels of each agent and pharmacological activity in vitro suggest very little difference in terms of immune enhancing properties. In contrast, Pom is a more potent anti-proliferative agent. However, it is unknown how MM cell sensitivity to Pom might be affected by resistance to Len or how sensitivity to Len might be affected by prior treatment with Pom. The use of a cellular model mimicking the relapse/resistance issue found in the clinic can provide valuable information directly translatable to the treatment of patients. MethodsIn this study we have generated MM cell resistance by long term exposure to either Len or Pom at levels attainable in patient plasma. H929 and KMS-12-BM MM cell lines were exposed to Len or Pom at clinically achievable concentrations (1 μM Len or 0.1 μM Pom) for a continuous period of 5 months. Cell growth was measured by ATP assay. Resistance factors were calculated by dividing the compound growth IC50 in the long term treated cells by the growth IC50 in the parental cells. ResultsThe continued culture of H929 and KMS-12-BM cells with Len resulted in Len resistance factors of >2270 and >47600, and Pom resistance factors of 725 and 280, respectively. Therefore, in the Len-resistant cells, cross-resistance to Pom was lower than resistance to Len. Conversely, the continued culture of H929 and KMS-12-BM cells with Pom resulted in Len resistance factors of >2270 and >47600, and Pom resistance factors of only 22 and 130, respectively. There was complete cross-resistance to Len in the Pom-resistant cells. Furthermore, the addition of Dex was able to improve the sensitivity of the resistant cells to Pom to levels that are attainable in the plasma of patients and far more than it improved sensitivity to Len. Thus, while Pom (and Pom/Dex) retained efficacy in Len-resistant MM cells, MM cells previously exposed to Pom became refractory to Len (and Len/Dex). ConclusionsRecent clinical data suggests that Pom/Dex is active in relapsed/refractory MM patients who have previously been treated with Len/Dex. Our results provide mechanistic support for the importance of the anti-proliferative activity of Pom (and Pom/Dex) in this setting. However, our results also suggest that Len (and Len/Dex) may be less effective in patients who have previously been treated with Pom, especially in the context of relapsed/refractory disease where immune function is likely to get progressively weaker with successive lines of therapy and thereby less amenable to immune enhancing agents. DisclosuresAdams:Celgene: Employment. Off Label Use: Pomalidomide is an anti-proliferative and immunomodulatory agent that is in clinical development for relapsed/refractory MM. Schafer:Celgene: Employment. Bartlett:Celgene: Employment.

Antiproliferative Effects of Lenalidomide in Combination with Chemotherapeutic Agents in Lymphoma Cell Lines

Introduction: Lenalidomide (Revlimid®) is approved for the treatment of patients with myelodysplastic syndromes with a del(5q), and in combination with dexamethasone for previously treated multiple myeloma. It is currently being evaluated in lymphomas, as monotherapy or combination therapy. This study aims to identify beneficial combinations of lenalidomide with established or potential chemotherapeutic agents for lymphoma therapy.Methods: Lenalidomide-sensitive lymphoma cell lines were selected for in vitro tumor cell proliferation studies. These included mantle cell lymphoma (MCL) lines Rec-1 and Jeko- 1, diffuse large-B-cell lymphoma (DLBCL) cell line Farage, the follicular lymphoma (FL) cell line DOHH2, and the chromosome 5-deleted Burkitt's lymphoma cell line Namalwa CSN.70. Lenalidomide was tested with other therapeutic agents on cells incubated 72 hours and assayed for proliferation using 3H-thymidine incorporation. Twenty-one different therapeutic agents were tested in combination with lenalidomide, at concentrations ranging from 1 pM to 100 μM, depending upon the sensitivity of the cells to the particular agent.Results: In MCL cells, lenalidomide was at least partially additive with mitoxantrone, rituximab, temsirolimus, bortezomib and azacytidine, and up to partially additive with doxorubicin. Results were similar in Rec-1 and Jeko-1 MCL cells. Lenalidomide was fully additive in the DLBCL cells with gemcitabine, melphalan, dexamethasone, carboplatin, and oxaliplatin, partially additive with cisplatin, mitoxantrone, vincristine, azacytidine, doxorubicin, carmustine, rituximab, etoposide and prednisolone, and non-additive with cytarabine. Synergy with lenalidomide was observed in the FL cells with rituximab, while partially additive with bendamustine and fludarabine, and non-additive with vincristine. In the Burkitt's lymphoma cells, lenalidomide was synergistic with dexamethasone, rapamycin, and UCN-01. It was partially additive with prednisone and etoposide, and non-additive with other agents tested.Conclusion: Among the tested lymphoma cell lines, lenalidomide combined synergistically with rituximab, dexamethasone, rapamycin, and UCN-01, and additively with mitoxantrone, temsirolimus, bortezomib, azacitidine, gemcitabine, melphalan, dexamethasone, carboplatin, and oxaliplatin. Thus, these results demonstrate a beneficial antineoplastic pharmacological interaction between lenalidomide and several therapeutic agents including monoclonal antibodies, corticosteroids, mTOR inhibitors, kinase inhibitors, proteasome inhibitors, DNA methyltransferase inhibitors, nucleoside analogs, alkylating agents, and topoisiomerase inhibitors in the treatment of lymphoma.

Lenalidomide Displays Direct Anti-Non-Hodgkin's Lymphoma (NHL) Cell Activity in Association with Enhanced SPARC Expression but Independent of Its Ability to Strongly Inhibit NHL Cell VEGF Production In Vitro.

Lenalidomide is an oral anti-angiogenic, anti-proliferative and immunomodulatory drug that is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk MDS associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone for the treatment of previously treated multiple myeloma patients. Encouraging early results suggest a potential for clinical efficacy in B cell non-Hodgkin's lymphoma (NHL). We have previously shown that lenalidomide can directly inhibit the proliferation of NHL cells and synergize with dexamethasone in inducing cell cycle arrest at the G1/S transition leading to caspase activation and apoptosis. In this study we have studied the potential for lenalidomide mediated growth inhibition, apoptosis and inhibition of angiogenic factors in NHL. We have assessed proliferation, expression of p21 and SPARC tumor suppressor genes and the production of the angiogenic factor vascular endothelial growth factor (VEGF) in six established NHL cells lines in vitro. The effect of lenalidomide on NHL cells was determined after 1–3 days using 3H-thymidine incorporation, microbead array and real time PCR. We found that lenalidomide displays anti-proliferative effects on Namalwa, Jeko-1 and Rec-1 cells when used alone and in combination with dexamethasone. However, several NHL cell lines, such as Granta-519, DB, and JVM-2, appear to be resistant. We also found that lenalidomide strongly inhibited VEGF production (but not mRNA levels) by the three sensitive lines (but not the three resistant lines) at much lower concentrations than required for inhibitory effects on proliferation. Excess recombinant human VEGF or neutralizing anti-VEGF antibody had no effect on Rec-1 proliferation suggesting that there is no connection between these two effects. Lenalidomide strongly increased the expression of tumor suppressor genes, such as p21cip/kip and SPARC (secreted protein acidic and rich in cysteine). In particular, elevation of SPARC mRNA correlated with the anti-proliferative effect of lenalidomide and was not seen in the resistant cells, whereas elevated p21 was observed in both sensitive and resistant (DB) cells. The effect of SPARC gene silencing on lenalidomide-induced effects is being assessed in siRNA studies. In conclusion, lenalidomide demonstrates anti-proliferative activity against certain sensitive NHL cells, particularly in combination with dexamethasone. Potent anti-VEGF activity of lenalidomide appears to be independent of proliferation and supports its anti-angiogenic potential. Lenalidomide-induced up-regulation of SPARC mRNA appears to correlate with sensitivity and may have biomarker potential in NHL.