Novel combination therapies with the RNA Polymerase I-mediated transcription inhibitor CX-5461 improve efficacy in the treatment of multiple myeloma

© 2019 Kylee Hannah MaclachlanMultiple myeloma (MM) is a malignant plasma cell disorder that is incurable with currently available therapy. The disease is genetically heterogeneous, with many recurrently mutated genes only seen in small numbers of patients and multiple clones present in each patient. This has limited potential approaches for designing widely applicable genetically targeted therapies. rDNA transcription is consistently dysregulated in cancer, mediated through both oncogenic and tumour-suppressive pathways. RNA polymerase I (Pol I) transcriptional hyperactivity is observed in many cancers, with this dysregulation shown to provoke a survival checkpoint in haematological tumour cells. With the hypothesis that the therapeutic targeting of Pol I transcription may prove an effective strategy across a variety of malignant settings, our laboratory co-developed CX-5461; a highly selective small molecule Pol I-mediated transcription inhibitor, now in phase 1 clinical trials in relapsed / refractory malignancies. We have previously demonstrated that single-agent treatment with CX-5461 provides a significant survival benefit in murine models of B-cell lymphoma and acute myeloid leukaemia. However, despite this improvement, drug resistance and relapse eventually occur, indicating combination drug therapy is essential for long term disease control and implementation in the clinic. This thesis examines combination drug strategies in MM, centred on the therapeutic inhibition of Pol I transcription of ribosomal genes, with the aim of accelerating the clinical use of CX-5461 for MM. A boutique, high-throughput screen in human myeloma cell lines (HMCLs) of CX-5461 in combination with drugs having known clinical or promising preclinical efficacy in MM revealed that CX-5461 increases anti-proliferative effects when combined with a range of other agents, encompassing various targets. The histone deacetylase inhibitor panobinostat and the proteasome inhibitor (PI) carfilzomib demonstrated the most impressive synergy in vitro, both representing drug classes that are actively used to treat patients with MM. In vivo testing demonstrated that the combination of CX-5461 with panobinostat increases survival compared with the single agents in both the Vκ*MYC murine model of MM and in C57BL-KaLwRij mice transplanted with 5T33 myeloma cells. Prolonged combination dosing in the Vκ*MYC model did not cause haematological toxicity beyond that seen with single agents. Investigating the molecular synergistic response to CX-5461 in combination with panobinostat indicated multiple potential mechanisms of synergy, including down-regulation of MYC and enhancement of the DDR elicited by CX-5461 alone. To extend the translation of CX-5461 and its combination with panobinostat into the clinic for MM, where resistance to front-line PI treatment frequently develops, we investigated the synergistic relationship of CX-5461 with each of these drug classes. In addition to the screen finding that CX-5461 synergised with each of panobinostat and carfilzomib, we showed the triplet was synergistic in vitro beyond the individual combinations. Moreover, modelling clinical PI resistance, we generated a cell line that is resistant to the front-line PI bortezomib, and demonstrated that CX-5461 retains its impressive efficacy in this setting, both in vitro and in vivo, using the 5T33-C57BL6/KaLwRij model. Taken together, the results described in this thesis will advance subsequent clinical trials utilising both CX-5461 and its combination with panobinostat in the treatment of relapsed multiple myeloma

Maintenance therapy and need for cessation studies in multiple myeloma: Focus on the future

With ten years of follow-up since the advent of the modern paradigm of combination induction therapy, consolidative autologous stem-cell transplant, and lenalidomide maintenance, median survival for multiple myeloma has increased to almost 50% at 10 years. Given this outlook, the overarching goal of maintenance therapy is to spare patients from the toxicities of aggressive or otherwise intrusive therapies while ideally extending survival or, at the least, extending progression-free survival or time until next treatment. This review will focus on the current landscape of maintenance therapies for multiple myeloma. The historical context and evidence for choice of agent, duration of treatment, and current strategies and ongoing trials will be discussed - as well as a focus on unmet needs. The case for studies investigating cessation of therapy and risk and response-adapted approaches will be underscored given that the current paradigm likely results in overtreatment for some patients

Novel combination therapy targeting rDNA transcription and Histone Deacetylation Provides Effective Treatment for Multiple Myeloma, and Synergises in Bortezomib-Resistant MM

Background: Multiple myeloma (MM) requires combination drug therapies to delay acquired drug resistance and clinical relapse. We co-developed CX-5461, a highly-selective inhibitor of RNA polymerase I-mediated rDNA transcription(1), currently in phase I trials for relapsed haematological malignancies (Peter Mac). CX-5461 produces a targeted nucleolar DNA damage response (DDR), triggering both a p53-dependent and -independent nucleolar stress response and killing malignant cells while sparing normal cells(2,3). Single-agent CX-5461 provides an impressive survival benefit in mouse models of B-cell lymphoma, acute myeloid leukaemia and now MM(2,4,5). However, drug resistance eventually occurs, confirming the need for combination therapies. Aim: To test the efficacy of CX-5461 in combination with the histone deacetylase inhibitor panobinostat, (prioritised from a boutique high-throughput screen of anti-myeloma agents), with a focus on the setting of resistance to proteasome-inhibitors (PIs). Methods: We assessed the impact of CX-5461 and panobinostat on overall survival in mouse models of MM, then surveyed the effects on cellular response and molecular markers of DDR. We developed bortezomib-resistant cell lines and an in vivo model of bortezomib-resistance to test this combination in the setting of PI-resistance. Results: CX-5461 in combination with panobinostat provides a significant survival advantage in both the transplanted Vk*MYC and the 5T33/KaLwRij models, with minimal bone marrow toxicity

Second malignancies in multiple myeloma; emerging patterns and future directions

The changing landscape of treatment options for multiple myeloma has led to a higher proportion of patients achieving deep, long-lasting responses to therapy. With the associated improvement in overall survival, the development of subsequent second malignancies has become of increased significance. The risk of second malignancy after multiple myeloma is affected by a combination of patient-, disease- and therapy-related risk factors. This review discusses recent data refining our knowledge of these contributing factors, including current treatment modalities which increase risk (i.e. high-dose melphalan with autologous stem cell transplant and lenalidomide maintenance therapy). We highlight emerging data towards individualized risk- and response-adapted treatment strategies and discuss key areas requiring future research

Defining the undetectable: The current landscape of minimal residual disease assessment in multiple myeloma and goals for future clarity

Multiple Myeloma, the second most prevalent hematologic malignancy, yet lacks an established curative therapy. However, overall response rate to modern four-drug regimens approaches 100%. Major efforts have thus focused on the measurement of minute quantities of residual disease (minimal residual disease or MRD) for prognostic metrics and therapeutic response evaluation. Currently, MRD is assessed by flow cytometry or by next generation sequencing to track tumor-specific immunoglobulin V(D)J rearrangements. These bone marrow-based methods can reach sensitivity thresholds of the identification of one neoplastic cell in 1,000,000 (10(-6)). New technologies are being developed to be used alone or in conjunction with established methods, including peripheral blood based assays, mass spectrometry, and targeted imaging. Data is also building for MRD as a surrogate endpoint for overall survival. Here, we will address the currently utilized MRD assays, challenges in validation across labs and clinical trials, techniques in development, and future directions for successful clinical application of MRD in multiple myeloma

Duration of Post-Autologous Hematopoietic Cell Transplant Anemia and Thrombocytopenia Are Associated with Prolonged Hospital Length-of-Stay for Multiple Myeloma Patients

BACKGROUND: Anemia, neutropenia, and thrombocytopenia are expected complications of autologous hematopoietic cell transplant (AHCT) for multiple myeloma (MM). However, prolonged cytopenias may predispose patients to other infectious, cardiovascular, and/or hematological toxicities. Various factors have been implicated in the length of post-AHCT cytopenias including stem cell dose, marrow microenvironment, and clonal hematopoiesis. We hypothesize that the length of post-transplant anemia, neutropenia, and thrombocytopenia may negatively impact hospital length-of-stay (LOS) and in-hospital complications. METHODS: This is a single-center observational study of MM patients who underwent AHCT. Patients were part of a larger cohort with detailed single nucleotide polymorphism (SNP) array cytogenetic data. Exclusion criteria were concurrent amyloidosis and tandem transplant. Demographic data, comorbidities, cytogenetic features (presence of TP53 deletion, and translocation status of MMSET, CCDN3, CCDN1, MAF, and MAFB), therapy line, peri-transplant laboratory values, and clinical outcome were collected retrospectively. LOS was calculated from transplant day -2 to hospital discharge. Predictive factors for LOS were calculated with multiple linear regression. Multiple logistic regression was then used to calculate associating factors for in-hospital complications. Grade III and IV post-transplant cardiovascular, infectious, and hematological complications were collected following the Common Terminology Criteria for Adverse Events (CTCAE). Post-transplant anemia was defined as sustained hemoglobin (Hb) <8 g/dL despite transfusions. The cutoffs for neutropenia and thrombocytopenia were absolute neutrophil count (ANC) <1.5 K/uL, and platelet (PLT) count <50 K/uL, respectively. RESULTS: 158 AHCT cases of MM patients were identified. 95 patients received AHCT as frontline treatment, 35 patients were transplanted in the salvage setting, and 14 patients received both frontline and subsequent-line transplant. The most commonly used conditioning regimen was melphalan 200 mg/m2 in 123 cases, followed by melphalan 140 mg/m2 in 23 patients. 50.6% (80/158) developed grade III anemia with a median onset of 9 days after transplant (IQR 5-11 days) and median length of 2 days (IQR 1-6 days). Neutropenia had higher incidence of 96.8% (153/158) with an earlier onset of 5 days (IQR 5-6 days) and median length of 5 days (IQR 2-7 days). Grade III thrombocytopenia occurred in 98.1% (155/158), with median onset of 7 days (IQR 5-7 days) and a median duration of 8 days (IQR 6-10 days). When comparing patients who received frontline transplant vs subsequent line transplants, no statistical significance was observed between onset or length of cytopenias (p=0.40). Median LOS was 16 days (IQR 15-19 days). The most frequent post-AHCT complications in our cohort were neutropenic fever (N=27), followed by engraftment syndrome (N=12), pneumonia (N=4), urinary tract infection (N=2), cellulitis (N=2), and bacteremia (N=1). Cardiovascular events were uncommon (N=3) and included pericarditis, new onset atrial fibrillation, and new onset supraventricular tachycardia. Pulmonary embolism (N=1) and deep vein thrombosis (N=3) were recorded. No major bleeding was observed. Longer LOS was independently associated with post-AHCT anemia (p=0.03) and thrombocytopenia (p=0.0005). Notably, LOS and in-hospital complication rates were not significantly associated with demographic data, pre-existing comorbidities, pre-transplant cytopenias, or cytogenetic abnormalities. CONCLUSION: In our cohort, longer duration of post-transplant anemia and thrombocytopenia were statistically associated with longer hospital LOS, but not with post-transplant complications. Prospective validation in an independent cohort is warranted. Disclosures Landgren: Karyopharma: Research Funding; Binding Site: Consultancy, Honoraria; Takeda: Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Janssen: Consultancy, Honoraria, Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Merck: Other; Seattle Genetics: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Adaptive: Consultancy, Honoraria; Juno: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Cellectis: Consultancy, Honoraria; Juno: Consultancy, Honoraria; Glenmark: Consultancy, Honoraria, Research Funding; Seattle Genetics: Research Funding; Pfizer: Consultancy, Honoraria; Merck: Other; Karyopharma: Research Funding; Binding Site: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Cellectis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Takeda: Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Glenmark: Consultancy, Honoraria, Research Funding. Chung:Genentech: Research Funding

Minimal residual disease in multiple myeloma: defining the role of next generation sequencing and flow cytometry in routine diagnostic use

For patients diagnosed with multiple myeloma (MM) there have been significant treatment advances over the past decade, reflected in an increasing proportion of patients achieving durable remissions. Clinical trials repeatedly demonstrate that achieving a deep response to therapy, with a bone marrow assessment proving negative for minimal residual disease (MRD), confers a significant survival advantage. To accurately assess for minute quantities of residual cancer requires highly sensitive methods; either multiparameter flow cytometry or next generation sequencing are currently recommended for MM response assessment. Under optimal conditions, these methods can detect one aberrant cell amongst 1,000,000 normal cells (a sensitivity of 10-6). Here, we will review the practical use of MRD assays in MM, including challenges in implementation for the routine diagnostic laboratory, standardisation across laboratories and clinical trials, the clinical integration of MRD status assessment into MM management and future directions for ongoing research