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Supplementary Fig. S1. Therapeutic efficacy of RV and HDACi against cancer B cells; Supplementary Fig. S2. HDACi increase JAM-1 expression without affecting cell viability; Supplementary Fig. S3. RV+HDACi display synergistic anti-myeloma activity; Supplementary Fig. S4. Combination treatment of MCL cells with RV and HDAC inhibitors results in increased cell death, compared to single agent treatments.

Elimination of drug-resistant leukemia stem cells (LSCs) represents a major challenge to achieve a cure in acute myeloid leukemia (AML). Although AML blasts generally retain high levels of surface CD38 (CD38pos), the presence of CD34 and lack of CD38 expression (CD34posCD38neg) are immunophenotypic features of both LSC-enriched AML blasts and normal hematopoietic stem cells (HSCs). We report that IFN-γ induces CD38 upregulation in LSC-enriched CD34posCD38negAML blasts, but not in CD34posCD38negHSCs. To leverage the IFN-γ mediated CD38 up-regulation in LSCs for clinical application, we created a compact, single-chain CD38-CD3-T cell engager (CD38-BIONIC) able to direct T cells against CD38posblasts. Activated CD4posand CD8posT cells not only kill AML blasts but also produce IFNγ, which leads to CD38 expression on CD34posCD38negLSC-enriched blasts. These cells then become CD38-BIONIC targets. The net result is an immune-mediated killing of both CD38negand CD38posAML blasts, which culminates in LSC depletion.Statement of significanceThis work represents a potential advancement in the treatment of AML, as it involves the release of IFN-γ by T cells to induce CD38 expression and thus sensitizing leukemia stem cells, which have been resistant to current treatment regimens, to CD38-directed T cell engagers.

Because most patients with multiple myeloma (MM) develop resistance to current regimens, novel approaches are needed. Genetically modified, replication-competent oncolytic viruses exhibit high tropism for tumor cells regardless of cancer stage and prior treatment. Receptors of oncolytic herpes simplex virus 1 (oHSV-1), NECTIN-1, and HVEM are expressed on MM cells, prompting us to investigate the use of oHSV-1 against MM. Using oHSV-1-expressing GFP, we found a dose-dependent increase in the GFP+ signal in MM cell lines and primary MM cells. Whereas NECTIN-1 expression is variable among MM cells, we discovered that HVEM is ubiquitously and highly expressed on all samples tested. Expression of HVEM was consistently higher on CD138+/CD38+ plasma cells than in non-plasma cells. HVEM blocking demonstrated the requirement of this receptor for infection. However, we observed that, although oHSV-1 could efficiently infect and kill all MM cell lines tested, no viral replication occurred. Instead, we identified that oHSV-1 induced MM cell apoptosis via caspase-3 cleavage. We further noted that oHSV-1 yielded a significant decrease in tumor volume in two mouse xenograft models. Therefore, oHSV-1 warrants exploration as a novel potentially effective treatment option in MM, and HVEM should be investigated as a possible therapeutic target., Graphical Abstract, Receptors of oncolytic herpes simplex virus (oHSV-1), NECTIN-1, and HVEM are expressed on multiple myeloma (MM) cells. Whereas NECTIN-1 expression is variable among MM cells, HVEM is ubiquitously and highly expressed on all samples tested. However, no viral replication occurred. Instead, oHSV-1 induced MM cell apoptosis via caspase-3 cleavage.

Reovirus is an oncolytic virus with natural tropism for cancer cells. We previously showed that reovirus intravenous administration in myeloma patients was safe, but disease control associated with viral replication in the cancer cells was not observed. Here we show that ex vivo proteasome inhibitors (PIs) potentiate reovirus replication in circulating classical monocytes, increasing viral delivery to myeloma cells. We found that the anti-viral signals in monocytes primarily rely on the NF-kB activation and that this effect is impaired by the addition of PIs. Conversely, PIs improved reovirus-induced monocyte and T cell activation against cancer cells. Based on these preclinical data, we conducted a phase 1b trial of the reovirus Pelareorep together with the PI carfilzomib in 13 heavily pretreated bortezomib-resistant MM patients. Objective responses associated with reovirus active replication in MM cells, T cell activation and monocytic expansion were noted in 70% of patients.

Simple Summary Targeted radionuclide therapy (TRT) and immunotherapy, an example being chimeric antigen receptor T cells (CAR-Ts), represent two potent means of eradicating systemic cancers. Although each one as a monotherapy might have a limited effect, the potency can be increased with a combination of the two therapies. The complications involved in the dosing and scheduling of these therapies make the mathematical modeling of these therapies a suitable solution for designing combination treatment approaches. Here, we investigate a mathematical model for TRT and CAR-T cell combination therapies. Through an analysis of the mathematical model, we find that the tumor proliferation rate is the most important factor affecting the scheduling of TRT and CAR-T cell treatments with faster proliferating tumors requiring a shorter interval between the two therapies. Abstract Targeted radionuclide therapy (TRT) has recently seen a surge in popularity with the use of radionuclides conjugated to small molecules and antibodies. Similarly, immunotherapy also has shown promising results, an example being chimeric antigen receptor T cell (CAR-T) therapy in hematologic malignancies. Moreover, TRT and CAR-T therapies possess unique features that require special consideration when determining how to dose as well as the timing and sequence of combination treatments including the distribution of the TRT dose in the body, the decay rate of the radionuclide, and the proliferation and persistence of the CAR-T cells. These characteristics complicate the additive or synergistic effects of combination therapies and warrant a mathematical treatment that includes these dynamics in relation to the proliferation and clearance rates of the target tumor cells. Here, we combine two previously published mathematical models to explore the effects of dose, timing, and sequencing of TRT and CAR-T cell-based therapies in a multiple myeloma setting. We find that, for a fixed TRT and CAR-T cell dose, the tumor proliferation rate is the most important parameter in determining the best timing of TRT and CAR-T therapies.

Key Points Teriflunomide, the active metabolite of leflunomide, downregulates c-Myc expression through inhibition of PIM kinases. Leflunomide together with lenalidomide significantly extended survival in an in vivo MM model.

Background: Immunotherapy with chimeric antigen receptor - T (CAR-T) cells and targeted radionuclide therapy (TRT) are two highly promising therapies in cancer treatment. Often, these therapies show limited efficacy in complete eradication of cancer cells making the combination of these two therapies an attractive cancer treatment option. The complications involved in dosing and scheduling of these therapies make mathematical modeling an appropriate method for analyzing and predicting disease response to these therapies. Here we propose a mathematical model evaluating disease response to the combination of these two therapies and explore the optimization of their dosing and scheduling. Methods: An ordinary differential equation-based formalism is proposed for simulation of tumor response to CAR-T cell therapy as well as TRT. CAR-T cell dose and injected radioactivity was input to the model. Among others, key model parameters included tumor proliferation rate, tumor cell and CAR-T cell radiosensitivity, CAR-T cell killing rate, CAR-T cell decay rate indicating persistence. Preclinical experiments involving CS1- CAR-T cell therapy and 225Ac-DOTA-Daratumumab TRT in a multiple myeloma mice model were used to parameterize the model. Sensitivity study of the model parameters using overall survival (OS) and progression-free survival (PFS) as evaluation metrics, was performed to elucidate the parameters with highest impact. Results: OS and PFS were 97 and 55 days when CAR-T cell therapy was given prior to TRT as compared to OS of 43 days for untreated control mice. Sensitivity study of model parameters showed that tumor proliferation has the highest impact on survival metrics. For a ±50% change in tumor proliferation rate, OS changed by -41%/+111% and PFS changed by -62%/+147%. Similar changes in TRT injected activity and CAR-T cell dose changed OS by ± 15% and ±21% respectively. Accordingly, PFS changed by roughly ±32% and ±45% respectively. A variation of the interval between the therapies showed that faster growing tumors required a shorter interval between the two therapies. The sequence of therapies was also changed and TRT prior to CAR-T cell therapy demonstrated shorter PFS (43 days) due to the adverse effects of radiation on CAR-T cells. Conclusion: For a fixed dose of TRT and CAR-T cells, tumor proliferation rate was found to be the prime factor impacting therapy interval. The presented work shows the key parameters required for planning and optimizing preclinical experiments and clinical trials. Using disease, CAR-T cell and radionuclide-specific parameters as shown in this work as well as incorporating immune stimulating effects of radiation would make it an extremely potent tool for optimizing combination therapies. Citation Format: Vikram Adhikarla, Dennis Awuah, Alexander B. Brummer, Enrico Caserta, Amrita Krishnan, Flavia Pichiorri, Megan M. Minnix, John E. Shively, Jeffrey Y.C. Wong, Xiuli Wang, Russell C. Rockne. A mathematical model for optimization of combination therapy involving targeted radionuclide and CAR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2732.

Multiple myeloma (MM) is characterized by an accumulation of malignant plasma cells (PCs) within the BM. The BM microenvironment supports survival of the malignant cells and is composed of cellular fractions that foster myeloma development and progression by suppression of the immune response. Despite major progress in understanding the biology and pathophysiology of MM, this disease is still incurable and requires aggressive treatment with significant side effects. CD84 is a self-binding immunoreceptor belonging to the signaling lymphocyte activation molecule (SLAM) family. Previously, we showed that CD84 bridges between chronic lymphocytic leukemia cells and their microenvironment, and it regulates T cell function. In the current study, we investigated the role of CD84 in MM. Our results show that MM cells express low levels of CD84. However, these cells secrete the cytokine macrophage migration inhibitory factor (MIF), which induces CD84 expression on cells in their microenvironment. Its activation leads to an elevation of expression of genes regulating differentiation to monocytic/granulocytic-myeloid-derived suppressor cells (M-MDSCs and G-MDSCs, respectively) and upregulation of PD-L1 expression on MDSCs, which together suppress T cell function. Downregulation of CD84 or its blocking reduce MDSC accumulation, resulting in elevated T cell activity and reduced tumor load. Our data suggest that CD84 might serve as a novel therapeutic target in MM.

Targeted therapies for multiple myeloma (MM) include the anti-CD38 antibody daratumumab, which, in addition to its inherent cytotoxicity, can be radiolabeled with tracers for imaging and with β- and α-emitter radionuclides for radioimmunotherapy. Methods: We have compared the potential therapeutic efficacy of β- versus α-emitter radioimmunotherapy using radiolabeled DOTA-daratumumab in a preclinical model of disseminated multiple myeloma. Multiple dose levels were investigated to find the dose with the highest efficacy and lowest toxicity. Results: In a dose–response study with the β-emitter (177)Lu-DOTA-daratumumab, the lowest tested dose, 1.85 MBq, extended survival from 37 to 47 d but did not delay tumor growth. Doses of 3.7 and 7.4 MBq extended survival to 55 and 58 d, respectively, causing a small equivalent delay in tumor growth, followed by regrowth. The higher dose, 11.1 MBq, eradicated the tumor but had no effect on survival compared with untreated controls, because of whole-body toxicity. In contrast, the α-emitter (225)Ac-DOTA-daratumumab had a dose-dependent effect, in which 0.925, 1.85, and 3.7 kBq increased survival, compared with untreated controls (35 d), to 47, 52, and 73 d, respectively, with a significant delay in tumor growth for all 3 doses. Higher doses of 11.1 and 22.2 kBq resulted in equivalent survival to 82 d but with significant whole-body toxicity. Parallel studies with untargeted (225)Ac-DOTA-trastuzumab conferred no improvement over untreated controls and resulted in whole-body toxicity. Conclusion: We conclude, and mathematic modeling confirms, that maximal biologic doses were achieved by targeted α-therapy and demonstrated (225)Ac to be superior to (177)Lu in delaying tumor growth and decreasing whole-body toxicity.

18F-Fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) is one of the most widely used imaging techniques to detect multiple myeloma (MM). Intracellular FDG uptake depicts in vivo metabolic activity, which can be seen in both malignant and nonmalignant cells, resulting in limited sensitivity and specificity. Our group showed preclinically that tracing MM dissemination using a CD38-directed human antibody, daratumumab, that is radioconjugated with 64Cu via the chelator DOTA (64Cu-daratumumab), led to improved sensitivity and specificity over that of FDG. Here, we report the results of a phase 1 trial designed to (1) assess the safety and feasibility of 64Cu-daratumumab PET/CT and (2) preliminarily evaluate and characterize the ability of 64Cu-daratumumab to accurately detect or exclude MM lesions. A total of 12 daratumumab-naive patients were imaged. Prior to the injection of 15 mCi/5 mg of 64Cu-daratumumab, patients were treated with 0 (n = 3), 10 (n = 3), 45 (n = 3), or 95 mg (n = 3) of unlabeled daratumumab to assess its effect on image quality. No significant adverse events were observed from either unlabeled daratumumab or 64Cu-daratumumab. Of the dose levels tested, 45 mg unlabeled daratumumab was the most optimal in terms of removing background signal without saturating target sites. 64Cu-daratumumab PET/CT provided safe whole-body imaging of MM. A trial comparing the sensitivity and specificity of 64Cu-daratumumab PET/CT with that of FDG PET/CT is planned. This trial was registered at www.clinicaltrials.gov as #NCT03311828.

Relating genotypes with phenotypes is important to understand diseases like cancer, but extremely challenging, given the underlying biological variability and levels of phenotypes. 3D quantitative tools are increasingly used to provide robust inferences pertaining to variations across collections of cells. We especially focus on the changes wrought to the nucleus of specific genotypes. Fibroblasts in the tumor microenvironment of mammary epithelial tissue serve as our model system and provide the context, although our methods are applicable to a broader range of biological systems. Using an image based approach, we analyze in 3D and compare phenotypes at nuclear level using estimates of texture, morphology and spatial context based on confocal images. Our data demonstrates that deletion of TP53 in stromal fibroblasts results in reorganization of chromatin content across the nucleus, especially the nuclear periphery, while simultaneously reducing nuclear size and making it more spindly. No such shape change was observed for PTEN-deleted genotype, although there were some differences in distribution of chromatin and an increase in the local nuclear density. The relative changes in phenotypes are in line with the larger role that the TP53 plays in tumor initiation and progression.These findings play an important role in uncovering the relationships of those genes with the subcellular phenotypes, as well as formulating new hypotheses, especially pertaining to the relative impact of genes in specific pathways. More importantly, they demonstrate the efficacy of methodology of analyzing a large number of cellular phenotypes.

The conserved Histidine 301 in switch II of Geobacillus stearothermophilus IF2 G2 domain was substituted with Ser, Gln, Arg, Leu and Tyr to generate mutants displaying different phenotypes. Overexpression of IF2H301S, IF2H301L and IF2H301Y in cells expressing wtIF2, unlike IF2H301Q and IF2H301R, caused a dominant lethal phenotype, inhibiting in vivo translation and drastically reducing cell viability. All mutants bound GTP but, except for IF2H301Q, were inactive in ribosome-dependent GTPase for different reasons. All mutants promoted 30S initiation complex (30S IC) formation with wild type (wt) efficiency but upon 30S IC association with the 50S subunit, the fMet-tRNA reacted with puromycin to different extents depending upon the IF2 mutant present in the complex (wtIF2 to IF2H301Q &gt, IF2H301R &gt, &gt, IF2H301S, IF2H301L and IF2H301Y) whereas only fMet-tRNA 30S-bound with IF2H301Q retained some ability to form initiation dipeptide fMet-Phe. Unlike wtIF2, all mutants, regardless of their ability to hydrolyze GTP, displayed higher affinity for the ribosome and failed to dissociate from the ribosomes upon 50S docking to 30S IC. We conclude that different amino acids substitutions of His301 cause different structural alterations of the factor, resulting in disparate phenotypes with no direct correlation existing between GTPase inactivation and IF2 failure to dissociate from ribosomes.

The conserved Histidine 301 in switch II of Geobacillus stearothermophilus IF2 G2 domain was substituted with Ser, Gln, Arg, Leu and Tyr to generate mutants displaying different phenotypes. Overexpression of IF2H301S, IF2H301L and IF2H301Y in cells expressing wtIF2, unlike IF2H301Q and IF2H301R, caused a dominant lethal phenotype, inhibiting in vivo translation and drastically reducing cell viability. All mutants bound GTP but, except for IF2H301Q, were inactive in ribosome-dependent GTPase for different reasons. All mutants promoted 30S initiation complex (30S IC) formation with wild type (wt) efficiency but upon 30S IC association with the 50S subunit, the fMet-tRNA reacted with puromycin to different extents depending upon the IF2 mutant present in the complex (wtIF2 ≥ to IF2H301Q > IF2H301R >>> IF2H301S, IF2H301L and IF2H301Y) whereas only fMet-tRNA 30S-bound with IF2H301Q retained some ability to form initiation dipeptide fMet-Phe. Unlike wtIF2, all mutants, regardless of their ability to hydrolyze GTP, displayed higher affinity for the ribosome and failed to dissociate from the ribosomes upon 50S docking to 30S IC. We conclude that different amino acids substitutions of His301 cause different structural alterations of the factor, resulting in disparate phenotypes with no direct correlation existing between GTPase inactivation and IF2 failure to dissociate from ribosomes.

INTRODUCTION: Although the treatment of patients with multiple myeloma (MM) has dramatically improved, those with high-risk characteristics, including the deletion or mutation of the master tumor suppressor gene TP53 on chromosome 17, experience limited survival. OM301 is a synthetic polypeptide containing the p53TA (transactivation) domain, which prevents p53 degradation through inhibition of MDM2. Here, we demonstrate that OM301 has strong anti-MM activity in vitro and in vivo. RESULTS: We first assessed the cytotoxic effects of OM301 in MM cell lines with varying TP53 status (TP53 wild type: MM.1S, H929; TP53 mutated/null: L363, RPMI-8226, U266, JJN3, KMS11) and found that OM301 exerts significant cytotoxic effects at a concentration of ~5 µM in all cell lines we tested, while it was minimally toxic to human peripheral blood mononuclear cells. Next, using immunocompromised NSG mice models injected with MM.1S, we determined the in vivo efficacy of OM301 in three different studies. Many potent anticancer agents, particularly of peptide origin, show prominent anti-tumor effects but fail to sustain similar effects when given intraperitoneally because of poor absorption, distribution, metabolism and excretion properties. OM301 at an intraperitoneal dose of 20 mg/kg/body weight twice a day induced significant reduction in tumor size with respect to vehicle control, suggesting the stability of OM301 without any loss of its activity (n=7, p Because OM301 was designed to simulate the p53 interaction domain with MDM2, we first determined its effect on p53-MDM2 crosstalk using a p53-MDM2 co-Immunoprecipitation (co-IP) assay and compared it with effects from Nutlin-3a, a known inhibitor of p53-MDM2. The co-IP data showed that, unlike Nutlin-3a, OM301 does not inhibit the p53-MDM2 interaction. Thus, to confirm our findings, we first overexpressed MDM2 in HeLa cells, and, using MDM2-IP and p53-MDM2 co-IP, found similar observations. Additionally, OM301 also failed to induce endogenous upregulation of genes activated by p53, such as MDM2 and p21, as opposed to results from Nutlin-3a. RNA sequencing data also showed a distinctive OM301signature, as compared to Nutlin-3a in MM cells. While treatment of Nutlin-3a induced expression of p53-activated canonical genes, OM301-treated cells showed alterations in genes involved in inflammatory responses, c-Myc regulated genes, fatty acid metabolism, glucose metabolism, and oxidative phosphorylation, among others. Next, to dissect its underlying mechanism, we dual-tagged OM301 with fluorophores at the 3' and 5' ends to study its localization and its stability in MM cells. Indeed, OM301 was found to be stable and mainly localized in the cytosol. We then modified OM301 by biotinylation of its penetratin end and first verified its cytotoxic effect in different MM cell lines, which was similar to that of native OM301. The biotinylated OM301 was then immunoprecipitated using streptavidin beads. The streptavidin pull-down and subsequent proteomic analysis confirmed that OM301 does not interact with MDM2 but interacts with c-Myc and with proteins localized in mitochondria, including Bcl-2 and Bcl-2 family members such as Bclaf1, Bcl2L13, and Bcl2L1. Pull-down experiments and immunoblot analysis validated Bcl-2/OM301 interactions. To further evaluate the relative binding potentials of OM301, we performed molecular docking studies using the HPEPDOCK server (Yan et al., Nat Protoc. 2020;15:1829). Post-docking, the calculated docking scores for OM301 was -281, suggesting that OM301directly interacts with Bcl-2. Thus, we evaluated the effects of OM301 on mitochondrial function and physiology. Treatment with OM301 decreased mitochondrial membrane potential in different MM cell lines. OM301 also increased mitochondrial superoxide production and induced mitophagy and mitochondrial fission as seen by electron microscopy. CONCLUSION: Here, we report for the first time that OM301, although designed for p53-selective cells, may instead interact with Bcl-2, which in turn induces mitochondrial dysfunction, leading to cell death irrespective of their TP53 status. Our data suggest that OM301 may be a novel and effective therapeutic option for MM. Figure 1 Figure 1. Disclosures Krishnan: REGENERON: Consultancy; MAGENTA: Consultancy; BMS: Consultancy, Current equity holder in publicly-traded company, Speakers Bureau; JANSSEN: Consultancy, Research Funding; City of Hope Cancer Center: Current Employment; SANOFI: Consultancy; GSK: Consultancy; Amgen: Speakers Bureau. Marcucci: Novartis: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings.

Daratumumab (Dara), a multiple myeloma (MM) therapy, is an antibody against the surface receptor CD38, which is expressed not only on plasma cells but also on NK cells and monocytes. Correlative data have highlighted the immune-modulatory role of Dara, despite the paradoxical observation that Dara regimens decrease the frequency of total NK cells. Here we show that, despite this reduction, NK cells play a pivotal role in Dara anti-MM activity. CD38 on NK cells is essential for Dara-induced immune modulation, and its expression is restricted to NK cells with effector function. We also show that Dara induces rapid CD38 protein degradation associated with NK cell activation, leaving an activated CD38-negative NK cell population. CD38+ NK cell targeting by Dara also promotes monocyte activation, inducing an increase in T cell costimulatory molecules (CD86/80) and enhancing anti-MM phagocytosis activity ex-vivo and in vivo. In support of Dara’s immunomodulating role, we show that MM patients that discontinued Dara therapy because of progression maintain targetable unmutated surface CD38 expression on their MM cells, but retain effector cells with impaired cellular immune function. In summary, we report that CD38+ NK cells may be an unexplored therapeutic target for priming the immune system of MM patients.

High levels of circulating miR-16 in the serum of multiple myeloma (MM) patients are independently associated with longer survival. Although the tumor suppressor function of intracellular miR-16 in MM plasma cells (PCs) has been elucidated, its extracellular role in maintaining a nonsupportive cancer microenvironment has not been fully explored. Here, we show that miR-16 is abundantly released by MM cells through extracellular vesicles (EVs) and that differences in its intracellular expression as associated with chromosome 13 deletion (Del13) are correlated to extracellular miR-16 levels. We also demonstrate that EVs isolated from MM patients and from the conditioned media of MM-PCs carrying Del13 more strongly differentiate circulating monocytes to M2-tumor supportive macrophages (TAMs), compared with MM-PCs without this chromosomal aberration. Mechanistically, our data show that miR-16 directly targets the IKKα/β complex of the NF-κB canonical pathway, which is critical not only in supporting MM cell growth, but also in polarizing macrophages toward an M2 phenotype. By using a miR-15a-16-1-KO mouse model, we found that loss of the miR-16 cluster supports polarization to M2 macrophages. Finally, we demonstrate the therapeutic benefit of miR-16 overexpression in potentiating the anti-MM activity by a proteasome inhibitor in the presence of MM-resident bone marrow TAM.

As a growing number of patients with multiple myeloma (MM) respond to upfront therapies while eventually relapsing in a time frame that is often unpredictable, attention has increasingly focused on developing novel diagnostic criteria to also account for disease dissemination. Positron emission tomography/computed tomography (PET/CT) is often used as a noninvasive monitoring strategy to assess cancer cell dissemination, but because the uptake of the currently used radiotracer 18fluorodeoxyglucose (

As a growing number of patients with multiple myeloma (MM) respond to upfront therapies while eventually relapsing in a time frame that is often unpredictable, attention has increasingly focused on developing novel diagnostic criteria to also account for disease dissemination. Positron emission tomography/computed tomography (PET/CT) is often used as a noninvasive monitoring strategy to assess cancer cell dissemination, but because the uptake of the currently used radiotracer 18fluorodeoxyglucose (18F-FDG) is a function of the metabolic activity of both malignant and nonmalignant cells, the results frequently lack sufficient specificity. Radiolabeled antibodies targeting MM tissue may detect disease irrespective of cell metabolism. Hence, we conjugated the clinically significant CD38-directed human antibody daratumumab (Darzalex [Dara]) to the DOTA chelator and labeled it with the positron-emitting radionuclide copper 64 (64Cu; 64Cu-DOTA-Dara). Here, we show that 64Cu-DOTA-Dara can efficiently bind CD38 on the surface of MM cells and was mainly detected in the bones associated with tumor in a MM murine model. We also show that PET/CT based on 64Cu-DOTA-Dara displays a higher resolution and specificity to detect MM cell dissemination than does 18F-FDG PET/CT and was even more sensitive than were bioluminescence signals. We therefore have supporting evidence for using 64Cu-DOTA-Dara as a novel imaging agent for MM.

Many amino acid-related genes in Gram-positive bacteria are regulated by the T box riboswitch. The leader RNA of genes in the T box family controls the expression of downstream genes by monitoring the aminoacylation status of the cognate tRNA. Previous studies identified a three-nucleotide codon, termed the "Specifier Sequence," in the riboswitch that corresponds to the amino acid identity of the downstream genes. Pairing of the Specifier Sequence with the anticodon of the cognate tRNA is the primary determinant of specific tRNA recognition. This interaction mimics codon-anticodon pairing in translation but occurs in the absence of the ribosome. The goal of the current study was to determine the effect of a full range of mismatches for comparison with codon recognition in translation. Mutations were individually introduced into the Specifier Sequence of the glyQS leader RNA and tRNA(Gly) anticodon to test the effect of all possible pairing combinations on tRNA binding affinity and antitermination efficiency. The functional role of the conserved purine 3' of the Specifier Sequence was also verifiedin this study. We found that substitutions at the Specifier Sequence resulted in reduced binding, the magnitude of which correlates well with the predicted stability of the RNA-RNA pairing. However, the tolerance for specific mismatches in antitermination was generally different from that during decoding, which reveals a unique tRNA recognition pattern in the T box antitermination system.

Acute reoviral infection has been extensively studied given the virus's propensity to target malignant cells and activate caspase-3 mediated apoptosis. Reovirus infection of malignant N1E-115 mouse neuroblastoma cells led to significant increased expression of importin-β and exportin-5 mRNAs (qRTPCR) and proteins (immunohistochemistry) which was partially blocked by small interfering LNA oligomers directed against the reoviral genome. Co-expression analysis showed that the N1E-115 cells that contained reoviral capsid protein had accumulated importin-β and exportin-5, as well as activated caspase 3. Reoviral oncolysis using a syngeneic mouse model of multiple myeloma similarly induced a significant increase in importin-β and exportin-5 proteins that were co-expressed with reoviral capsid protein and caspase-3. Apoptotic proteins (BAD, BIM, PUMA, NOXA, BAK, BAX) were increased with infection and co-localized with reoviral capsid protein. Surprisingly the anti-apoptotic MCL1 and bcl2 were also increased and co-localized with the capsid protein suggesting that it was the balance of pro-apoptotic molecules that correlated with activation of caspase-3. In summary, productive reoviral infection is strongly correlated with elevated importin-β and exportin-5 levels which may serve as biomarkers of the disease in clinical specimens.

Oncolytic viruses (OVs) are emerging as powerful anti-cancer agents and are currently being tested for their safety and efficacy in patients. Reovirus (Reolysin), a naturally occurring non-pathogenic, double-stranded RNA virus, has natural oncolytic activity and is being tested in phase I–III clinical trials in a variety of tumor types. With its recent US Food and Drug Administration (FDA) orphan drug designation for several tumor types, Reolysin is a potential therapeutic agent for various cancers, including head and neck squamous cell carcinomas (HNSCCs), which have a 5-year survival of ∼55%. Histone deacetylase inhibitors (HDACis) comprise a structurally diverse class of compounds with targeted anti-cancer effects. The first FDA-approved HDACi, vorinostat (suberoylanilide hydroxamic acid [SAHA]), is currently being tested in patients with head and neck cancer. Recent findings indicate that HDAC inhibition in myeloma cells results in the upregulation of the Reolysin entry receptor, junctional adhesion molecule 1 (JAM-1), facilitating reovirus infection and tumor cell killing both in vitro and in vivo. In this study, we tested the anti-tumor efficacy of HDAC inhibitors AR-42 or SAHA in conjunction with Reolysin in HNSCCs. While HDAC inhibition increased JAM-1 and reovirus entry, the impact of this combination therapy was tested on the development of anti-tumor immune responses.

Introduction: 18Fluoro-deoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) is one of the most widely used imaging techniques to detect multiple myeloma (MM). Intracellular FDG uptake depicts in vivo metabolic activity, which can be seen in both malignant and non-malignant cells, resulting in limited sensitivity and specificity. Our group showed preclinically that tracing MM dissemination using a CD38-directed human antibody, daratumumab, that is radioconjugated with copper-64 via the chelator DOTA (64Cu-DOTA-Dara), led to improved sensitivity and specificity over that of FDG (Caserta et al, Blood 2018). Herein, we report the results of a Phase 1 trial (NCT#03311828) designed to 1) assess the safety and feasibility of 64Cu-DOTA-Dara PET/CT and 2) to evaluate and characterize the ability of 64Cu-DOTA-Dara to accurately detect or exclude MM lesions compared with FDG PET/CT. Methods: Patients with biopsy-proven MM and/or a plasmacytoma received an FDG PET/CT scan within 60 days of enrollment. On Day 0, patients were infused with unlabeled ("cold") Dara at one of four dose levels (0 mg, 10 mg, 45 mg, 95 mg) to optimize biodistribution of radioconjugated 64Cu-DOTA-Dara, especially in the liver and the spleen. Within 6 hours of unlabeled Dara administration, patients received 64Cu-DOTA-Dara at a dose of 13.63-16.68 mCi (~5 mg). Whole-body PET scans were obtained at 24 hours and at 48 hours (the latter scan encompassing known tumors). 64Cu-DOTA-Dara standardized uptake values (SUV) were evaluated in MM lesions and normal organs, which were then compared with values from standard FDG PET/CT. Biopsies were performed on accessible discordant lesions. Results: A total of 10 Dara-naïve patients were imaged. Patients were treated with 0 (n=3), 10 (n=3), 45 (n=3), or 95 mg (n=1) of unlabeled Dara. Four patients had newly diagnosed disease, one had biochemical relapse, one had a recurrent plasmacytoma by MRI, and four had possible recurrence by standard PET/CT. No significant adverse events were observed from either cold or 64Cu-DOTA-Dara. With the exception of the one patient with a recurrent plasmacytoma, radiolabeled antibody was eliminated from systemic circulation in subjects analyzed at the first three dose levels within 30 min post injection. In the patient with a recurrent plasmacytoma, the radiolabeled antibody was elevated in the blood for over two days. One newly diagnosed patient had extensive disease by FDG PET and had a biopsy-proven target lesion in the sternum, which had an SUVmax of 14.7 on 64Cu-DOTA-Dara PET/CT vs. 3.3 onFDG PET/CT. A second biopsy from the same patient was taken from a discordant iliac crest lesion (positive for 64Cu-DOTA-Dara but negative for FDG PET/CT) that showed 20-30% MM cell infiltration. In another patient, an iliac crest lesion was 64Cu-DOTA-Dara positive and FDG-negative; biopsy revealed 6% plasmacytosis in the bone. A third patient had an FDG PET/CT positive pleural lesion with an SUVmax of 8.98 and negative on 64Cu-DOTA-Dara (Figure 1A). The lesion did not show recurrence upon biopsy. Furthermore, 64Cu-DOTA-Dara PET/CT yielded superior imaging of bone lesions in the calvarium (Figure 1B). Escalating doses of unlabeled Dara decreased liver and spleen uptake by 64Cu-DOTA-Dara. Conclusions: In this ongoing study, 64Cu-DOTA-Dara PET/CT imaging is to date safe and provides whole body imaging of MM. Further dose escalation of cold Dara (145 mg, 195 mg) is planned to optimize background interference. This modality has the potential to improve sensitivity and specificity over FDG PET/CT scanning in early-stage MM as well as in recurrent disease. Disclosures Krishnan: Celgene, Janssen, Sanofi, BMS: Consultancy; Sutro BioPharma, zPredicta: Consultancy; Amgen, Takeda: Speakers Bureau; Celgene, Z Predicta: Other: Stock Ownership; Takeda: Research Funding. Palmer:Gilead Sciences: Consultancy. Rosenzweig:Celgene, Takeda: Speakers Bureau. Wu:ImaginAb, Inc.: Consultancy, Other: Board Member.

INTRODUCTION: Pelareorep is the infusible form of human reovirus (RV). Our single-agent phase 1 RV trial in relapsed multiple myeloma (MM) showed that pelareorep treatment selectively infected MM cells, as viral RNA was found in myeloma cells and not the bone marrow (BM) stroma. However, we did not observe apoptosis. Our ongoing phase 1 trial, which combines the proteasome inhibitor carfilzomib with RV, has demonstrated RV infection, apoptosis, and clinical responses. We investigated the molecular mechanisms behind the role of a PI (carfilzomib) in this setting. RESULTS: In all MM cell lines we tested (n=4), independently from their sensitivity to RV infection (Stiff et al., Mol Cancer Ther, 2016), viral replication and apoptosis was impaired when MM cells were directly exposed to PIs (carfilzomib and bortezomib). When this experiment was repeated in the setting of the bone marrow cellular fraction or peripheral blood mononuclear cells (PBMCs), it had the opposite effect, as the addition of PI to RV increased RV replication and apoptosis in MM cells. When we washed PBMCs after overnight exposure to RV+PI or to either single agent, then added MM cells, we observed higher infection and apoptosis in cancer cells co-cultured with RV+PI compared to levels from PBMCs treated with each of the single agents, suggesting that PIs increase the ability of PBMCs to serve as a reservoir for infectious reovirus. Monocytes (CD14+) can engage in phagocytosis of reovirus (Berkeley et al., Cancer Immunol Res, 2018), and accordingly we found that RV genome and capsid protein production were detected in CD14+ cells, but not in CD14-depleted PBMCs, and increased upon PI treatment compared to that in RV-treated CD14+ cells. Given that the NF-κB complex is a key proinflammatory signaling pathway associated with the early innate-antiviral immune response, and because PIs can block the degradation of the NF-κB inhibitor IκBα upon phosphorylation, we investigated the effect of the specific IκBα inhibitor Bay-11 in RV viral replication. Our data show that either PI or Bay-11 can inhibit RV-induced IκBα phosphorylation and its subsequent degradation upon RV infection in CD14+ cells, an effect associated with higher capsid formation in RV-treated CD14+ cells in combination with PI or Bay-11, compared to levels from RV alone. Cytokine profiling in PBMCs and CD14+ cells treated with RV in combination with either PI or Bay-11 showed a significant decrease in IFN-α and IFN-β (IFNs) levels and a concomitant increase in RV replication, in contrast to levels from RV alone (p We then decided to investigate whether CD14 depletion could affect RV delivery to the cancer cells invivo. Upon intra-femoral injection of 5TGM1 MM cells into syngeneic C57BL/KaLwRij mice, the mice in which the monocytes were depleted by clodronate-liposome treatment before intravenous RV injection showed lower capsid protein formation in the BM MM cells compared to that in mice where the monocytic population was intact. Because monocytes respond to infection by dividing into macrophages to eliminate pathogens, we wanted to investigate whether PI could impair this effect. Intriguingly, although higher levels of viral genome were detected in PI+RV-treated CD14+ cells compared to RV-treated cells (p CONCLUSIONS: Here we report for the first time that PIs enhance pelareorep entry, infection, and killing of myeloma cells through its effect on the CD14+ fraction. Reovirus infection and replication within CD14+ cells are augmented by PI-induced NF-κB inhibition of the early innate pro-inflammatory immune response. We also report for the first time that carfilzomib induces direct T cell activation and potentiates T cell killing activity against RV-infected MM cells. Disclosures Krishnan: Takeda: Research Funding; Celgene, Z Predicta: Other: Stock Ownership; Amgen, Takeda: Speakers Bureau; Sutro BioPharma, zPredicta: Consultancy; Celgene, Janssen, Sanofi, BMS: Consultancy. Sborov:Celgene: Honoraria; Janssen: Consultancy. Hofmeister:Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imbrium: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees.

Multiple myeloma remains incurable and the majority of patients die within 5 years of diagnosis. Reolysin, the infusible form of human reovirus (RV), is a novel viral oncolytic therapy associated with antitumor activity likely resulting from direct oncolysis and a virus-mediated antitumor immune response. Results from our phase I clinical trial investigating single agent Reolysin in patients with relapsed multiple myeloma confirmed tolerability, but no objective responses were evident, likely because the virus selectively entered the multiple myeloma cells but did not actively replicate. To date, the precise mechanisms underlying the RV infectious life cycle and its ability to induce oncolysis in patients with multiple myeloma remain unknown. Here, we report that junctional adhesion molecule 1 (JAM-1), the cellular receptor for RV, is epigenetically regulated in multiple myeloma cells. Treatment of multiple myeloma cells with clinically relevant histone deacetylase inhibitors (HDACi) results in increased JAM-1 expression as well as increased histone acetylation and RNA polymerase II recruitment to its promoter. Furthermore, our data indicate that the combination of Reolysin with HDACi, potentiates RV killing activity of multiple myeloma cells in vitro and in vivo. This study provides the molecular basis to use these agents as therapeutic tools to increase the efficacy of RV therapy in multiple myeloma. Mol Cancer Ther; 15(5); 830–41. ©2016 AACR.

Expression of conjugative transfer and virulence functions of the Enterococcus faecalis antibiotic resistance plasmid pCF10 is regulated by the interaction of the pheromone receptor protein PrgX with two DNA binding operator sites (XBS1 and XBS2) upstream from the transcription start site of the prgQ operon (encoding the pCF10 transfer machinery) and by posttranscriptional mechanisms. Occupancy of both binding sites by PrgX dimers results in repression of the prgQ promoter. Structural and genetic studies suggest that the peptide pheromone cCF10 functions by binding to PrgX and altering its oligomerization state, resulting in reduced occupancy of XBSs and increased prgQ transcription. The DNA binding activity of PrgX has additional indirect regulatory effects on prgQ transcript levels related to the position of the convergently transcribed prgX operon. This has complicated interpretation of previous analyses of the control of prgQ expression by PrgX. We report here the results of in vivo and in vitro experiments examining the direct effects of PrgX on transcription from the prgQ promoter, as well as quantitative correlation between the concentrations of XBSs, PrgX protein, and prgQ promoter activity in vivo . The results of electrophoretic mobility shift assays and quantitative analysis of prgQ transcription in vitro and in vivo support the predicted roles of the PrgX DNA binding sites in prgQ transcription regulation. The results also suggest the existence of other factors that impede PrgX repression or enhance its antagonism by cCF10 in vivo .

Introduction: Multiple Myeloma (MM) cell survival strictly depends on the interaction with multiple cell types in the bone marrow (BM), collectively referred to as the MM microenvironment (MM-ME). CD84 (SLAMF5) belongs to the signaling lymphocyte activation molecule family of immunoreceptors; previous data from our group have shown that CD84 mediates malignant B cells and their ME (Marom et al. 2017); however, its role within the MM-ME has not yet been investigated. Results: Using the MMRF CoMMpass IA9 dataset, we found that CD84 mRNA expression is low or absent in CD138+MM cells isolated from 660 newly diagnosed patients. In agreement with these data, flow analysis showed an absence of CD84 expression in all MM cell lines tested (n=9) and minimal surface expression (5.5-13%) in primary cells (n=3). However, a significantly higher expression of CD84 was detected on the surface of BM and peripheral blood (PB) monocytic fractions (CD14+) (76-97%) compared to that of matched CD14 negative fractions (2-9%) obtained from 16 different MM patients (p Next, to understand whether the expansion of Mo-MDSC and associated CD84 up-regulation is directly dependent on the MM cells, we investigated Mo-MDSC expansion and CD84 expression in two different MM mouse models. Specifically, 5TGM1 murine MM cells were IV injected into syngeneic immune-competent KaLwRijHsd mice (n=7), and human MM.1S cells were IV injected into immune-deficient NSG mice (n=10). Our data show that MM progression leads to significant Mo-MDSC expansion (p Since T cell checkpoint inhibitors PD-1/PD-L1 are tumor immune escape receptors that play a pivotal role in supporting an immunosuppressive MM-ME, we also investigated the expression of PD-L1 on the CD14+ fraction in MM patients. BM-CD14+ cells (n=3) were compared with matched CD14 negative cells. We found that the CD14+ fraction had a higher PD-L1 expression in the BM of MM patients compared to the CD14 negative fraction (60-96% versus 3-8%). Hence, we investigated whether CD84-mediated cell-cell contact may increase the level of PD-L1 expression on Mo-MDSCs and whether MM cells can regulate CD84 and PD-L1 expression on CD14+ cells. Co-culture assays were performed using several human MM cell lines (MM1S, U266 and KMS11) with PB CD14+ cells derived from healthy donors (n=3). Our data show a 1.9-fold increase from the basal level of CD84 (27±16% to 50±9.4%) and a 2.9-fold increase from the basal level of PD-L1 (18±8.5% to 52.3±5.7%). Our recently generated mouse anti-human CD84 blocking antibody (Marom et al. 2017) was used to determine whether CD84 direct targeting can affect MM-induced CD84 and PD-L1 expression on the surface of CD14+cells. Co-culture experiments show that targeting CD84 lowered CD84 (1.6-fold decrease) and PD-L1 expression (1.8-fold decrease) on a CD14+ population co-cultured with MM cells. Moreover, when T cells were included in the co-culture experiments, we observed reduced expression of exhaustion markers PD-1 (1.1-fold decrease) and CTLA4 (1.4-fold decrease) in the presence of the CD84 blocking antibody. Conclusion: We show here that CD84 is expressed on Mo-MDSCs in MM and that the expansion of this myeloid population is strictly associated with MM progression. Disruption of CD84 contacts with a blocking antibody decreases the expression of PD-L1 on CD14+ cells and exhaustion markers on T cells. Thus, our results reveal a novel role for CD84 in regulating PD-L1 in the MM-ME and provides the scientific rationale to investigate whether targeting CD84 expression on Mo-MDSCs can restore T cell functions. To further confirm the role of CD84 regulating T cell activity and Mo-MDSC, in vivo mouse studies are ongoing and results will be presented at the meeting. Disclosures Rosenzweig: Celgene: Speakers Bureau. Krishnan:Sutro: Speakers Bureau; Onyx: Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Equity Ownership, Speakers Bureau; Takeda: Speakers Bureau.

Daratumumab (Dara), a human immunoglobulin G1 kappa (IgG1κ) monoclonal anti-CD38 antibody, has been approved by the U.S. Food and Drug Administration for the treatment of relapsed multiple myeloma (MM) as a single agent as well as in combination with immunomodulatory drugs (IMiDs) and proteasome inhibitors (PI). Although the scientific rationale behind the use of Dara in combination with IMiDs has been extensively explored, the molecular mechanisms underlying Dara-PI regimens have not yet been investigated. Here, we demonstrate that CD38 on the surface of MM cells is rapidly internalized after Dara treatment; we also show that Dara treatment impairs MM cell adhesion, an effect that can be rescued by using the endocytosis inhibitor Dynasore. Finally, we show that Dara potentiates bortezomib (BTZ) killing of MM cells in vitro and in vivo, independent of its function as an immune activator. In conclusion, our data show that Dara impairs MM cell adhesion, which results in an increased sensitivity of MM to proteasome inhibition.

Germline mutations in the tumor suppressor gene PTEN (phosphatase and tensin homology deleted on chromosome 10) cause Cowden and Bannayan–Riley–Ruvalcaba (BRR) syndromes, two dominantly inherited disorders characterized by mental retardation, multiple hamartomas, and variable cancer risk. Here, we modeled three sentinel mutant alleles of PTEN identified in patients with Cowden syndrome and show that the nonsense Pten ∆4–5 and missense Pten C124R and Pten G129E alleles lacking lipid phosphatase activity cause similar developmental abnormalities but distinct tumor spectra with varying severity and age of onset. Allele-specific differences may be accounted for by loss of function for Pten ∆4–5 , hypomorphic function for Pten C124R , and gain of function for Pten G129E . These data demonstrate that the variable tumor phenotypes observed in patients with Cowden and BRR syndromes can be attributed to specific mutations in PTEN that alter protein function through distinct mechanisms.

InfB-encoded translation initiation factor IF2 contains a non-conserved N-terminal domain and two conserved domains (G and C) constituted by three (G1, G2 and G3) and two (C1 and C2) sub-domains. Here, we show that: (i) Bacillus stearothermophilus IF2 complements in vivo an Escherichia coli infB null mutation and (ii) the N-domain of B. stearothermophilus IF2, like that of E. coli IF2, provides a strong yet dispensable interaction with 30 S and 50 S subunits in spite of the lack of any size, sequence or structural homology between the N-domains of the two factors. Furthermore, the nature of the B. stearothermophilus IF2 sites involved in establishing the functional interactions with the ribosome was investigated by generating deletion, random and site-directed mutations within sub-domains G2 or G3 of a molecule carrying an H301Y substitution in switch II of the G2 module, which impairs the ribosome-dependent GTPase activity of IF2. By selecting suppressors of the dominant-lethal phenotype caused by the H301Y substitution, three independent mutants impaired in ribosome binding were identified; namely, S387P (in G2) and G420E and E424K (in G3). The functional properties of these mutants and those of the deletion mutants are compatible with the premise that IF2 interacts with 30 S and 50 S subunits via G3 and G2 modules, respectively. However, beyond this generalization, because the mutation in G2 resulted in a functional alteration of G3 and vice versa, our results indicate the existence of extensive "cross-talking" between these two modules, highlighting a harmonic conformational cooperation between G2 and G3 required for a functional interaction between IF2 and the two ribosomal subunits. It is noteworthy that the E424K mutant, which completely lacks GTPase activity, displays IF2 wild-type capacity in supporting initiation of dipeptide formation.

The tumour stroma is believed to contribute to some of the most malignant characteristics of epithelial tumours. However, signalling between stromal and tumour cells is complex and remains poorly understood. Here we show that the genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumours. This was associated with the massive remodelling of the extracellular matrix (ECM), innate immune cell infiltration and increased angiogenesis. Loss of Pten in stromal fibroblasts led to increased expression, phosphorylation (T72) and recruitment of Ets2 to target promoters known to be involved in these processes. Remarkably, Ets2 inactivation in Pten stroma-deleted tumours ameliorated disruption of the tumour microenvironment and was sufficient to decrease tumour growth and progression. Global gene expression profiling of mammary stromal cells identified a Pten-specific signature that was highly represented in the tumour stroma of patients with breast cancer. These findings identify the Pten-Ets2 axis as a critical stroma-specific signalling pathway that suppresses mammary epithelial tumours.

Inactivation of phosphatase and tensin homology deleted on chromosome 10 (PTEN) is linked to increased PI3K–AKT signaling, enhanced organismal growth, and cancer development. Here we generated and analyzed Pten knock-in mice harboring a C2 domain missense mutation at phenylalanine 341 (PtenFV), found in human cancer. Despite having reduced levels of PTEN protein, homozygous PtenFV/FV embryos have intact AKT signaling, develop normally, and are carried to term. Heterozygous PtenFV/+ mice develop carcinoma in the thymus, stomach, adrenal medulla, and mammary gland but not in other organs typically sensitive to Pten deficiency, including the thyroid, prostate, and uterus. Progression to carcinoma in sensitive organs ensues in the absence of overt AKT activation. Carcinoma in the uterus, a cancer-resistant organ, requires a second clonal event associated with the spontaneous activation of AKT and downstream signaling. In summary, this PTEN noncatalytic missense mutation exposes a core tumor suppressor function distinct from inhibition of canonical AKT signaling that predisposes to organ-selective cancer development in vivo.

Multiple myeloma (MM) is a hematological malignancy of plasma cells in the bone marrow. Despite multiple treatment options, MM is inevitably associated with drug resistance and poor outcomes. Histone deacetylase inhibitors (HDACi's) are promising novel chemotherapeutics undergoing evaluation in clinical trials for the potential treatment of patients with MM. Although in preclinical studies HDACi's have proven anti-myeloma activity, but in the clinic single-agent HDACi treatments have been limited due to low tolerability. Improved clinical outcomes were reported only when HDACi's were combined with other drugs. Here, we show that a novel pan-HDACi AR-42 downregulates CD44, a glycoprotein that has been associated with lenalidomide and dexamethasone resistance in myeloma both in vitro and in vivo. We also show that this CD44 downregulation is in part mediated by miR-9-5p, targeting insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which directly binds to CD44 mRNA and increases its stability. Importantly, we also demonstrate that AR-42 enhances anti-myeloma activity of lenalidomide in primary MM cells isolated from lenalidomide resistant patients and in in vivo MM mouse model. Thus, our findings shed light on potential novel combinatorial therapeutic approaches modulating CD44 expression, which may help overcome lenalidomide resistance in myeloma patients.

Many amino acid-related genes in Gram-positive bacteria are regulated by the T box riboswitch. The leader RNA of genes in the T box family controls the expression of downstream genes by monitoring the aminoacylation status of the cognate tRNA. Previous studies identified a three-nucleotide codon, termed the "Specifier Sequence," in the riboswitch that corresponds to the amino acid identity of the downstream genes. Pairing of the Specifier Sequence with the anticodon of the cognate tRNA is the primary determinant of specific tRNA recognition. This interaction mimics codon-anticodon pairing in translation but occurs in the absence of the ribosome. The goal of the current study was to determine the effect of a full range of mismatches for comparison with codon recognition in translation. Mutations were individually introduced into the Specifier Sequence of the glyQS leader RNA and tRNA(Gly) anticodon to test the effect of all possible pairing combinations on tRNA binding affinity and antitermination efficiency. The functional role of the conserved purine 3' of the Specifier Sequence was also verifiedin this study. We found that substitutions at the Specifier Sequence resulted in reduced binding, the magnitude of which correlates well with the predicted stability of the RNA-RNA pairing. However, the tolerance for specific mismatches in antitermination was generally different from that during decoding, which reveals a unique tRNA recognition pattern in the T box antitermination system.

Bacterial translation initiation factor IF2 is a GTP-binding protein that catalyzes binding of initiator fMet-tRNA in the ribosomal P site. The topographical localization of IF2 on the ribosomal subunits, a prerequisite for understanding the mechanism of initiation complex formation, has remained elusive. Here, we present a model for the positioning of IF2 in the 70S initiation complex as determined by cleavage of rRNA by the chemical nucleases Cu(II):1,10-orthophenanthroline and Fe(II):EDTA tethered to cysteine residues introduced into IF2. Two specific amino acids in the GII domain of IF2 are in proximity to helices H3, H4, H17, and H18 of 16S rRNA. Furthermore, the junction of the C-1 and C-2 domains is in proximity to H89 and the thiostrepton region of 23S rRNA. The docking is further constrained by the requisite proximity of the C-2 domain with P-site-bound tRNA and by the conserved GI domain of the IF2 with the large subunit’s factor-binding center. Comparison of our present findings with previous data further suggests that the IF2 orientation on the 30S subunit changes during the transition from the 30S to 70S initiation complex.