Wang, Demin

Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells (PCs) and aberrant production of monoclonal immunoglobulin detected as an M spike using serum protein electrophoresis. In the United States, MM represents ~15% of hematologic malignancies and is one of the few cancers increasing in incidence (e.g., 14,400 in 1996 to 32,270 in 2020, from SEER). Despite recent treatment advances, patients diagnosed with high risk/refractory MM (hrrMM) have an overall survival of only three years. Recently, our lab developed the first genetically engineered NrasQ61R; Myc-driven myeloma model (VQ mice). The VQ model recapitulates most biological and clinical features of hrrMM. VQ cells are serially transplantable into syngeneic recipients and can be propagated in vitro. As such, the VQ model could serve as a robust pre-clinical platform for drug screening and in vivo testing of new therapies to treat hrrMM. Whole exome sequencing (WES) of CD138+ cells from five primary VQ mice identified eight recurrently mutated genes. Of these eight genes, two have been previously identified as recurrently mutated in MM patients while the remainder appear to be unique to VQ myeloma. Meanwhile, RNA sequencing of CD138+ cells taken from both primary VQ mice and secondary VQ recipients show differential gene expression for five of these eight genes. Gene ontology analysis of recurrently mutated genes indicates that these genes play a role in interferon signaling, as well as receptor tyrosine kinase signaling. Here, we report the results of a high-throughput repurposing screen using the NCI's AOD IX library, which consists of 147 FDA-approved anti-cancer drugs. VQ cells were treated with 0.1 and 1µM of each compound in the presence or absence of 10nM of Trametinib (Tra), a potent MEK inhibitor. Screening results returned expected positive hits that were previously FDA approved for MM treatment (green dots, Panel 1B), such as proteasome inhibitors, histone deacetylase inhibitors, and chemotherapy drugs. Importantly, VQ cells were resistant to some compounds currently used to treat MM, including the BCL-2 inhibitor Venetoclax, the alkylating agent Melphalan, and the topoisomerase II inhibitor Etoposide (red dots, Panel 1B). De novo resistance to these therapies further indicates that the VQ cells model a multi-drug resistant MM. Two compounds were chosen for further testing: the protein translation inhibitor Omacetaxine (Oma) based on its single agent efficacy, and the multi-receptor tyrosine kinase inhibitor Ponatinib (Pot) based on its high synergy with Tra. Combination Index testing confirmed initial drug screening results but showed some antagonism between combination Oma and Pot treatment. We proceeded with in vivo testing of two novel combination therapies, in which 3-week treatment cycles were followed by a 1-week rest period between cycles: Three weeks of daily Tra and Pot treatment (TP), or two weeks of Tra and Pot treatment followed by one week of Oma (TPO) treatment. Vehicle (Veh) treated and Tra only treated mice were used as controls. Treatment efficacy was assessed against VQ-D1 MM, which grows primarily in the bone marrow (BM) and metastasizes to the spleen and liver of recipient mice. Four weeks post-treatment, myeloma growth in the BM was significantly reduced in both combo treatment groups, while MM burden in the spleen was also reduced. Overall, both TP and TPO treatment significantly extended survival compared to Veh or Tra treated mice, and significantly reduced liver extramedullary disease. To further test the range of Tra/Pot synergy in vivo, Tra dosage was reduced and treatment was administered daily. Interestingly, both low dose Tra monotherapy and TP combo therapy significantly extended survival compared to Veh control or Pot alone, and TP treated mice had increased survival compared to the Tra only treated mice. Taken together, our results show that low dose daily Trametinib treatment is superior to cycling dosage, and that Ponatinib treatment synergizes with both dosing schedules to extend survival in vivo. Our study demonstrates the utility of the VQ model for identifying new treatments for hrrMM and testing their efficacy in vivo. As part of our ongoing drug repurposing experiments, we have expanded our screening to a panel of 2,580 FDA-approved drugs, including but not limited to anti-cancer and anti-inflammatory drugs. Figure Disclosures Callander: Cellectar: Research Funding; University of Wisconsin: Current Employment.

Heparin-induced thrombocytopenia (HIT) is a serious reaction to heparin treatment characterized by antibodies that recognize a complex formed between heparin and platelet factor 4 (PF4/H) and are capable of activating platelets and inducing a pro-thrombotic state. Although a high percentage of heparin-treated patients produce antibodies to PF4/H, only a subset of these antibodies are platelet-activating (pathogenic) and capable of causing HIT. We previously reported that we cloned B cells from six patients experiencing HIT and identified two types of PF4/H-binding antibodies: seven platelet-activating (PA) and 48 non-activating (NA). Comparison of the structural features in the PA, NA, and non PF4/H-binding (NB) clones showed that the length and the number of basic amino acid and tyrosine residue in the heavy chain complementarity determining region 3 (HCDR3) were significantly different, and was in the order of PA>NA>NB. Most significantly, the seven platelet-activating antibodies each have one of the two pathogenic motifs: RX1-2 R/KX1-2 R/H and YYYYY in an unusually long HCDR3 (≥ 20 residues). In the current study, we attempt to understand the origin of the B cells that produce the PA and NA antibodies and the nature of the immune response in HIT through analyzing somatic hypermutation and biological property of such antibodies. Longer HCDR3 and more basic Aas and Tyr residues in the HCDR3 are features of autoreactive and polyreactive antibodies. With this in mind, we tested PA and NA clones in a standard antinuclear antibody (ANA) assay and found that these clones were significantly more reactive than NB antibodies, and the plasma of HIT patients were significantly more reactive than normal plasma (Figure1). We then compared reactions of PA, NA and NB clones against a group of self and foreign antigens commonly used in polyreactivity assays: dsDNA, ssDNA, LPS, insulin, and keyhole limpet hemocyanin (KLH). About 90% of PA and NA clones were reactive to at least two antigens, this was true of only 20% of the NB clones, and the latter is consistent with the frequency of polyreactive clones in the IgG+ B cells (Figure2). Taken together, these data indicate that PA and NA antibodies are largely polyreactive. We then investigated the development of the PA and NA B cells through analyzing somatic hypermutation in the antibodies. Through analyzing the HCDR3 nucleotide insertion, trimming and VDJ segment usage, we found that longer HCDR3 typical of PF4/H-binding clones and the RKH and Y5 motifs identified in PA clones were the result of original recombination not somatic hypermutation. Consistently, the average number of nucleotide mutations in the VH genes of the binding clones was lower (PA and NA, 9.4 ± 9.5) compared to that of peripheral blood IgG+ memory B cells in healthy subjects (~18) (Figure3). Total mutation frequency in the VH and Vk CDRs of the PF4/H-binding PA and NA clones was comparable to that of the framework regions. This finding contrasts with findings made in peripheral blood IgG+ memory B cells of healthy subjects showing that the mutation frequencies are much higher in the CDRs than in the FRs of VH. Taken together, these findings suggest that affinity maturation plays a limited role in the evolution PF4/H-binding antibodies during the immune response that leads to HIT. In this study, we showed thay PF4/H-binding PA and NA IgGs are largely polyreactive antibodies and contain lower levels of mutations compared to IgG+ memory B cells. B1 and MZ B cells are innate B cells that are main producers of polyreactive natural antibodies and can respond to toll-like receptor signaling, quickly differentiate into antibody-secreting cells, and undergo IgG class switch extrafollicularly. Polyreactivity identified in the PF4/H-binding PA and NA IgGs supports the possibility that human B cells producing PF4/H-binding antibodies are innate B cells akin to MZ B cells shown to be a source of PF4/H antibodies in mice. A mutation rate lower than that of IgG+ memory cells in the PF4/H-binding IgGs is also consistent with an extrafollicular response typical of innate B cells. These observations would help to improve our understanding of the immunological responses and B cell origin in HIT patients. Disclosures Padmanabhan: Retham Technologies: Current equity holder in private company; Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees; Versiti Blood Research Institute: Patents & Royalties.

Abstract NRAS Q61 mutations are prevalent in advanced/relapsed multiple myeloma (MM) and correlate with poor patient outcomes. Thus, we generated a novel MM model by conditionally activating expression of endogenous NrasQ61R and an MYC transgene in germinal center (GC) B cells (VQ mice). VQ mice developed a highly malignant MM characterized by a high proliferation index, hyperactivation of extracellular signal-regulated kinase and AKT signaling, impaired hematopoiesis, widespread extramedullary disease, bone lesions, kidney abnormalities, preserved programmed cell death protein 1 and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain immune-checkpoint pathways, and expression of human high-risk MM gene signatures. VQ MM mice recapitulate most of the biological and clinical features of human advanced/high-risk MM. These MM phenotypes are serially transplantable in syngeneic recipients. Two MM cell lines were also derived to facilitate future genetic manipulations. Combination therapies based on MEK inhibition significantly prolonged the survival of VQ mice with advanced-stage MM. Our study provides a strong rationale to develop MEK inhibition–based therapies for treating advanced/relapsed MM.

Abstract Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells (PCs) and aberrant production of monoclonal immunoglobulin detected as an M spike using serum protein electrophoresis. In the United States, MM represents ~15% of hematologic malignancies and is one of the few cancers increasing in incidence (e.g., 14,400 in 1996 to 30,770 in 2018, from SEER). Previously, a Vk*MYC mouse model was described, in which AID-dependent activation of MYC transgene in germinal center (GC) B cells catalyzes a highly penetrant, indolent MM after a prolonged latency, suggesting that additional genetic mutations are required for the malignant MM progression. Recent sequencing of paired tumor/normal samples from advanced or refractory MM patients identified that constitutive activation of Ras signaling pathway (KRAS: 23%; NRAS:20%; BRAF: 8%) associates with MM progression and therapy resistance. To determine whether oncogenic Nras promotes the progression of Myc-induced indolent MM to a malignant stage, we generated NrasLSL Q61R/+; Vk*MYC; IgG1-Cre (VQ) mice along with single mutant mice Vk*MYC; IgG1-Cre or NrasLSL Q61R/+; IgG1-Cre. To boost NrasQ61R expression in GC B cells, 6-7 weeks old mice were immunized with NP-CGG. A significant fraction of VQ mice developed M-spike after immunization and subsequently died of a highly aggressive MM, which was characterized by high proliferative index, hyperactivation of AKT and ERK pathways, and disease hallmarks (e.g. osteolytic lesions, anemia, and kidney injury). VQ myeloma was readily transplantable into serial syngeneic recipients. In our preliminary study, combined Bortezomib and AZD6244 treatment attenuated MM phenotypes and prolonged the survival of VQ recipients. To facilitate easy molecular or genetic manipulations of VQ MM cells, we established two cell lines from primary cells. These cells could be easily infected (see another abstract from our co-author, Dr. Asimakopoulos's group). The cultured MM cells also express high levels of CD155, a ligand of immune checkpoint TIGIT, which has been recently reported to mediate a significant immune checkpoint blockade in MM patients. We further investigated the transcriptional signature of VQ MM cells (from bone marrow [BM] and from lymph nodes [LN]) vs wild-type (WT) plasma cells using the Illumina Bio-Rad's Single-Cell Sequencing platform. Illumina's Sure Cell app was used to de-multiplex samples, process barcodes and perform single-cell 3' gene counting. The processed data were then analyzed using Seurat R package to yield cluster information and their associated enriched genes. Our results revealed distinct clusters of CD138+ MM and WT cells, and also showed high similarity & overlap between BM & LN MM samples. Consistent with the genetic changes introduced to VQ MM cells, both Myc and Nras expression levels were significantly elevated in MM clusters compared to WT clusters. We also found that transcriptional levels of several potential therapeutic targets were significantly elevated in MM clusters, including Integrin alpha 4 and metabolic enzymes. Currently we are validating the scRNA-Seq results. Taken together, we generated a novel mouse model in which activation of Vk*MYC and oncogenic Nras in GC B cells results in a highly malignant, transplantable MM. The VQ mouse model represents an important innovation that will serve as a platform to investigate pathogenesis of resistant and refractory multiple myeloma and allow for testing the efficacy of novel therapeutic agents. Disclosures No relevant conflicts of interest to declare.

Abstract Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells (PCs) and aberrant production of monoclonal immunoglobulin detected as an M spike using serum protein electrophoresis. In the United States, MM represents ~15% of hematologic malignancies and is one of the few cancers increasing in incidence (e.g., 14,400 in 1996 to 30,330 in 2016, from SEER). Previously, a Vk*MYC mouse model was described, in which AID-dependent activation of MYC transgene in germinal center (GC) B cells catalyzes a highly penetrant, indolent MM after a prolonged latency. However, the indolent MM developed in Vk*MYC mice rarely progresses to a malignant stage, suggesting that additional genetic mutations are required for the malignant MM progression. Recent sequencing of paired tumor/normal samples from advanced or refractory MM patients identified that constitutive activation of Ras signaling pathway (KRAS: 23%; NRAS:20%; BRAF: 8%) associates with MM progression and therapy resistance. To determine whether oncogenic Nras promotes the progression of Myc-induced indolent MM to a malignant stage, we generated NrasLSL Q61R/+; Vk*MYC; IgG1-Cre (VQ) mice along with single mutant mice Vk*MYC; IgG1-Cre (Vk) or NrasLSL Q61R/+; IgG1-Cre (Q61R). To boost NrasQ61R expression in GC B cells, 6-7 weeks old mice were immunized with NP-CGG. A significant fraction of VQ mice developed M-spike after immunization and subsequently died of a highly malignant MM. The MM developed in some mice were restricted in bone marrow and spleen and thus mimicked majority of MM patients, while MM developed in other mice was primarily located in lymph nodes and represented patients with extramedullary MM. Both types of MM cells displayed hyperproliferation and hyperactivation of AKT and ERK pathways. They could be cultured for several weeks with their original stromal cells and in the presence of Il-6. The cultured MM cells could be easily infected or transfected. Upon transplantation, both types of MM cells caused a rapid lethality (within 16 and 4 weeks respectively) in secondary and tertiary recipients and preserved the initial locations of donor MM cells. MM recipient mice developed bone lesions and/or fractures characteristic of MM patients. Preliminary preclinical studies indicated that both types of MM cells were resistant to Bortezomib, a proteasome inhibitor commonly used in treating MM patients, and AZD6244, a potent MEK inhibitor. Additional tests of combinatorial therapies are ongoing. Taken together, we generated a novel mouse model in which activation of Vk*MYC and oncogenic Nras in GC B cells results in a highly malignant, transplantable MM. This model can serve as a platform to investigate pathogenesis of highly malignant multiple myeloma and test the efficacy of novel therapeutic agents. Citation Format: Zhi Wen, Adhithi Rajagopalan, Erik A. Ranheim, Remington Finn, Adam Pagenkopf, Grant Yun, Yun Zhou, Yidan Wang, Demin Wang, Marta Chesi, P. Leif Bergsagel, Fotis Asimakopoulos, Jing Zhang. Expression of oncogenic Nras and a MYC transgene in germinal center B cells induces a highly malignant multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3018.

AbstractCREB-binding protein (CBP) and its para-log p300 are transcriptional coactivators that physically or functionally interact with over 320 mammalian and viral proteins, including 36 that are essential for B cells in mice. CBP and p300 are generally considered limiting for transcription, yet their roles in adult cell lineages are largely unknown since homozygous null mutations in either gene or compound heterozygosity cause early embryonic lethality in mice. We tested the hypotheses that CBP and p300 are limiting and that each has unique properties in B cells, by using mice with Cre/LoxP conditional knockout alleles for CBP (CBPflox) and p300 (p300flox), which carry CD19Cre that initiates floxed gene recombination at the pro–B-cell stage. CD19Cre-mediated loss of CBP or p300 led to surprisingly modest deficits in B-cell numbers, whereas inactivation of both genes was not tolerated by peripheral B cells. There was a moderate decrease in B-cell receptor (BCR)–responsive gene expression in CBP or p300 homozygous null B cells, suggesting that CBP and p300 are essential for this signaling pathway that is crucial for B-cell homeostasis. These results indicate that individually CBP and p300 are partially limiting beyond the pro-B-cell stage and that other coactivators in B cells cannot replace their combined loss.