Multiple Myeloma (MM) is a malignancy of plasma cells that affects over 30,000 Americans every year. Despite advances in the treatment of the disease, approximately 12,000 American patients will still die of MM in 2019. One of the mainstays of treatment for MM is the immunomodulatory and antiangiogenic drug lenalidomide; which is used in induction therapy, maintenance therapy and treatment of relapsed disease. Although not fully elucidated, lenalidomide's mechanism of action in MM involves the drug binding to Cerebelon (CBN) and leads to the subsequent degradation of the Ikaros (IKZF1) and Aiolos (IKZF3) transcription factors (TF). These TFs play important regulatory roles in lymphocyte development. Despite lenalidomide's importance in MM treatment, several groups have reported that MM patients treated with lenalidomide rarely go on to develop B-cell acute lymphoblastic leukemia (B-ALL). The genetics and clonal relationship between the MM and subsequent B-ALL have not been previously defined. Importantly, it is not clear if the MM and B-ALL arise from the same founding clone that has been under selective pressure during lenalidomide treatment. As deletions in IKZF1 are common in B-ALL, one could hypothesize that lenalidomide's mechanism of action mimics this alteration and contributes to leukemogenesis.
We sequenced the tumors from a cohort of seven patients with MM treated with lenalidomide who later developed B-ALL. These data did not show any mutational overlap between the MM and ALL samples-the tumors arose from different founding clones in each case. However, several genes were recurrently mutated in the B-ALL samples across the seven patients. These genes included TP53, ZFP36L2, KIR3DL2, RNASE-L, and TERT. Strikingly, five of the seven patients had a TP53 mutations in the B-ALL sample that was not present in the matched MM sample. The frequency of TP53 mutations in our cohort was much higher than that reported in adult de novo B-ALL patients which can range between 4.1-6.4% (Hernández-Rivas et al. 2017 and Foa et al. 2013). Utilizing CRISPR-Cas9 gene editing, we disrupted the Zfp36l2 or Actb in murine hematopoietic stem cells (HSCs) of mice with or without loss of Trp53. We performed our first transplantation experiment in which the cohorts of mice have loss of Trp53 alone, loss of Zfp36l2 alone, loss of both Trp53 and Zfp36l2, or a control knockout (KO) of Actb. To characterize the disruption of Zfp36l2 alone and in combination with Trp53 we analyzed the hematopoietic stem and progenitor cell compartments in the bone marrow of the above transplanted mice. In mice with a loss of Zfp36l2 there is a decrease in Lin- Sca-1+ c-Kit+ (LSK), short term-HSC (ST-HSC), and multipotent progenitors (MPP). This decrease was not observed in the mice with a loss of both Trp53 and Zfp36l2, where instead we noted an increase in monocyte progenitors (MP), granulocytes-macrophage progenitors (GMP), and common myeloid progenitors (CMP) cells. In this Trp53 Zfp36l2 double loss model we also noted a decrease in B220+ B-cells that was not seen in the Zfp36l2 alone. In this cohort of Trp53 Zfp36l2 loss, we characterized B-cell development through hardy fraction flow cytometry, and identified a decrease in fractions A and B/C (pre-pro and pro-B-cells, respectively) as compared to Zfp36l2 or Actb alone.
As lenalidomide does not bind to Cbn in mice, we used the human B-ALL NALM6 cell line to test if treatment with lenalidomide will lead to a selective growth advantage of cells with the same genes knocked out versus wild-type control cells grown in the same culture. We hypothesize that lenalidomide treatment selectively enriched for pre-existing mutated cell clones that evolved into the B-ALL. Preliminary data in NALM6 cells with a loss of TP53 demonstrate a slight increase in cell number at day 7 compared to a RELA control. These experiments will be repeated with concurrent ZFP36L2 and TP53 mutations as well as ZFP36L2 alone. Treatment-related disease is a key consideration when deciding between different treatment options, and this project aims to understand the relationship between MM treatment and B-ALL occurrence. It may be possible to identify MM patients who are at-risk for B-ALL. For example, MM patients who harbor low-level TP53 mutations prior to lenalidomide treatment could be offered alternative treatment options.
Barnell: Geneoscopy Inc: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Wartman:Novartis: Consultancy; Incyte: Consultancy.
Pomalidomide selectively targets BCL11A and SOX6 to induce γ-globin synthesis. The mechanism of action of pomalidomide during erythropoiesis is independent of IKZF1 degradation, in contrast to multiple myeloma.
Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment such as cytokines and growth factors, nutrients and stresses to regulate multiple cellular processes, including translation, autophagy, metabolism, growth, motility and survival. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 and TORC2. Activation of TORC1 leads to the phosphorylation of p70S6 kinase and 4E-BP1, while activation of TORC2 regulates phosphorylation of Akt and other AGC kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin analogues such as RAD001 and CCI-779 have been tested in clinical trials in MM. Their efficacy as single agents is modest, but when used in combination, they show higher responses. However, total inhibition of Akt and 4E-BP1 signaling requires inactivation of both complexes TORC1 and TORC2. Consequently, there is a need for novel inhibitors that can target mTOR in both signaling complexes. In this study we have evaluated the role of TORC1 and TORC2 in MM and the activity and mechanism of action of INK128, a novel, potent, selective and orally active small molecule TORC1/2 kinase inhibitor.
Nine different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. Nanofluidic proteomic immunoassays were performed in selected tumors.
To examine activation of the mTOR pathway in MM, we performed kinase activity assays and protein analyses of mTOR complexes and its downstream targets in nine MM cell lines. We found mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all cell lines tested independently of the status of Deptor, PTEN, and PI3K. All cell lines expressed either Raptor, Rictor or both; excepting H929 and U266LR7 which were negative for both of them. Moreover, primary plasma cells from several MM patients highly expressed pS6R while normal cells were negative for this protein. We found that INK128 and rapamycin effectively suppressed phosphorylation of p6SR, but only INK128 was able to decrease phosphorylation of 4E-BP1. We observed that INK128 fully suppressed cell viability in a dose and time dependent manner, but rapamycin reached a plateau in efficacy at ± 60%. The IC50 of INK128 was in the range of 7.5–30 nM in the eight cell lines tested. Similar results were observed in freshly isolated plasma cells from MM patients. Besides the induction of apoptosis and cell cycle arrest, INK128 was more potent than rapamycin to induce autophagy, and only INK128 was able to induce PARP and Caspases 3, 8 and 9 cleavage. In the bone marrow microenvironment context, INK128 inhibited the proliferation of MM cells and decreased the p4E-BP1 induction. Importantly, treatment with rapamycin under such conditions did not affect cell proliferation. INK128 also showed a significantly greater effect inhibiting cell adhesion to fibronectin OPM2 MM1S, BMSCs and HUVECs compared to rapamycin. These results were confirmed in vivo. Oral daily treatment of NK128 (1.0 mg/kg) decreased tumor growth and improved survival of mice implanted with MM1S.
Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients.
Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.
Approximately 50% of the Multiple Myeloma (MM) cases are associated with hyperdiploidy (Hyperdiploid MM, HDMM), usually of the odd numbered chromosomes. The majority of the remaining patients with MM have translocations involving the immunoglobulin heavy chain (IgH) locus on chromosome 14q32 (Translocated MM, TMM).(Fonseca R, Leukemia 2009;23:2210-21)
However, most of these estimates are from studies done in predominantly white populations. MM is approximately two fold more common in blacks, and results from the phase 3 study of lenalidomide and dexamethasone suggested that blacks may have better outcome. (Rajkumar SV, Lancet Oncol 2010;11(1):29-37)
The purpose of this study was to determine the cytogenetic characteristics and outcome of MM in blacks, and to compare this with a similar population of white patients seen during the same time period.
We studied African American and African patients with MM diagnosed from May 2002 through June 2010 at John H. Stroger, Jr. Hospital of Cook County (CC), Chicago, IL and comparable Caucasian population seen at Mayo Clinic (MC), Rochester, MN between these dates with available cytogenetic studies (karyotyping and/or fluorescent in situ hybridization (FISH) data) at diagnosis. The results between both populations were then compared.
65 blacks (25 men and 40 women) and 490 Caucasians (290 men and 200 women) with MM were studied. Karyotyping was available in 98% of patients from both cohorts, and FISH was available for 35% and 64% of the CC and MC cohorts, respectively. A molecular classification was possible in 16 (25%) of the CC cohort (12 HDMM, 4 TMM) and 294 (60%) of the MC cohort (38% HDMM, 22% TMM). In the remaining patients, both tests were normal or showed changes not definitive for either IgH translocations or hyperdiploidy. At diagnosis, abnormal cytogenetics by karyotyping was present in 25/65 (38%) of the CC cohort and 154/479 (32%) of the MC cohort. The median overall survival (OS) from diagnosis for the CC and MC cohorts was similar at 64 months. The median OS was significantly shorter for those with an abnormal cytogenetics; 35 months vs. 65 months in the CC cohort (P0.03) and 49 months vs. not reached for the MC cohort (P < 0.01). OS was superior among patients in the CC cohort initially treated with an immunomodulatory agent (IMiD) containing regimen (65 months vs. 35 months for those receiving a bortezomib or other non-IMiD containing regimens (P=0.02; Figure 1). Similarly, in the MC cohort, patients receiving an IMiD based regimen had a superior OS (not reached vs. 42 months; P < 0.001). The use of immunomodulatory therapy did not differ across cytogenetic types.
The current study suggests that IgH translocations were less frequently seen in blacks compared with what is typically reported in the literature. This may explain the relatively better survival reported in blacks compared with whites in recent trials. We found a significantly superior outcome in blacks treated initially with regimens containing immunomodulatory agents compared to other regimens.
No relevant conflicts of interest to declare.
Constitutive and immunoproteasome inhibitors (C&IPI) were thought to suppress nuclear factor-κB (NF-κB) pathway by preventing IκB degradation, which prevents NF-κB translocation into the nucleus. This mechanism of action has since been questioned by a number of studies. First, bortezomib promoted constitutive NF-κB activity in endothelial cell carcinoma. Second, NF-κB constitutive activity was resistant to bortezomib in multiple myeloma cell lines. Third, bortezomib increased IκB mRNA but post-transcriptionally downregulated IκB in normal cells and in multiple myeloma cell lines resulting in induced canonical NF-κB activation. Lastly, bortezomib increased nuclear levels of IκB as opposed to lowering cytoplasmic levels in cutaneous T cell lymphoma cell line suggesting that nuclear translocation of IκB was possibly responsible for NF-κB inhibition. The inhibitory activity of C&IPI on dendritic cells (DC) is of interest in the prevention of graft versus host disease (GvHD). It has been shown that different C&IPI impede DC maturation and T cell priming both in vitro and in vivo. Herein we sought to understand the mechanism of action of proteasome and immunoproteasome inhibitors on DC and to test their effect on IκB and NF-IκB expression.
Materials and Methods
We first performed RT PCR on lysates of DC obtained from the peripheral blood of 7 patients who received post-transplant cyclophosphamide and bortezomib as prevention of GvHD on a phase I clinical trial. Patients received allogeneic transplantation from matched-related or unrelated donors. Patients received no other immunosuppressive therapy except for rabbit anti-thymocyte globulin for those receiving graft from unrelated donor. Steroids were not allowed on the study. Samples were obtained on days +1, +4, and +7. The results were analyzed in comparison to samples obtained on day 0 before stem cell infusion. We then performed the same experiment on lysates of DC obtained from the peripheral blood of healthy volunteer donors. DC were untreated or incubated with bortezomib (10 nM for 4 h), carfilzomib (30 nM for 1 h), oprozomib (100 nM and 300 nM for 4 h), ONX 0914 (200 nM for 1 h), PR-825 (125 nM for 1 h), or PR-924 (1000 nM for 1 h). The drug concentration and duration of exposure were chosen based on the IC50 on proteasome activity and to reproduce in vivo conditions. We also performed IκB western blot on DC isolated from peripheral blood of healthy volunteers, untreated or incubated with bortezomib (10 nM for 4 h) or oprozomib (300 nM for 4 h). Each experiment was performed at least in triplicate.
We found that the combination of cyclophosphamide and bortezomib significantly and progressively increased IκB mRNA while decreasing NF-κB mRNA in DC studied ex vivo. We also found that all studied C&IPI increased IκB mRNA to a variable degree while only oprozomib (300 nM) decreased NF-κB mRNA in DC in vitro. Finally, both bortezomib and oprozomib increased IκB protein level in DC in vitro (figure).
Our data suggest that C&IPI increase IκB expression in DC. As opposed to the previously reported data in other cell types, the effect is not associated with post-transcriptional downregulation. Cyclophosphamide and bortezomib also decrease NF-κB expression in DC in vivo while only oprozomib had the same effect in vitro. The effect of C&IPI on IκB and NF-κB expression may represent a new mechanism of action and suggests their effect may be cell-type dependent.
Al-Homsi: Millennium Pharmaceuticals: Research Funding. Off Label Use: The use of cyclophosphamide and bortezomib for GvHD prevention. Lai:Millennium Pharmaceuticals: Research Funding.