A synthetically lethal precision medicine therapy for HK1-HK2+ multiple myeloma using an HK2 antisense oligonucleotide, metformin, and perhexiline. Most normal tissues Tolerated Tolerated HK1 + HK2 + multiple myeloma HK 2-A SO Me tfo rmi n Per hex ilin e HK1-HK2 + multiple myeloma Glucose-6-P Glucose-6-P Glucose Glucose HK1 HK2 HK2 Synthetically lethal Although the majority of adult tissues express only hexokinase 1 (HK1) for glycolysis, most cancers express hexokinase 2 (HK2) and many coexpress HK1 and HK2. In contrast to HK1 þ HK2 þ cancers, HK1 À HK2 þ cancer subsets are sensitive to cytostasis induced by HK2 shRNA knockdown and are also sensitive to synthetic lethality in response to the combination of HK2 shRNA knockdown, an oxidative phosphorylation (OXPHOS) inhibitor diphenyleneiodonium (DPI), and a fatty acid oxidation (FAO) inhibitor perhexiline (PER). The majority of human multiple myeloma cell lines are HK1 À HK2 þ. Here we describe an antisense oligonucleotide (ASO) directed against human HK2 (HK2-ASO1), which suppressed HK2 expression in human multiple myeloma cell cultures and human multiple myeloma mouse xenograft models. The HK2-ASO1/DPI/PER triple-combination achieved synthetic lethality in multiple myeloma cells in culture and prevented HK1 À HK2 þ multiple myeloma tumor xenograft progression. DPI was replaceable by the FDA-approved OXPHOS inhibitor metformin (MET), both for synthetic lethality in culture and for inhibition of tumor xenograft progression. In addition, we used an ASO targeting murine HK2 (mHK2-ASO1) to validate the safety of mHK2-ASO1/MET/PER combination therapy in mice bearing murine multiple myeloma tumors. HK2-ASO1 is the first agent that shows selective HK2 inhibition and therapeutic efficacy in cell culture and in animal models, supporting clinical development of this synthetically lethal combination as a therapy for HK1 À HK2 þ multiple myeloma. Significance: A first-in-class HK2 antisense oligonucleotide suppresses HK2 expression in cell culture and in in vivo, presenting an effective, tolerated combination therapy for preventing progression of HK1 À HK2 þ multiple myeloma tumors.

Multiple myeloma (MM), a plasma cell malignancy, is the second most prevalent hematologic malignancy in the US. Although much effort has been made trying to understand the etiology and the complexities of this disease with the hope of developing effective therapies, MM remains incurable at this time. Because of their antiproliferative and proapoptotic activities, interferons (IFNs) have been used to treat various malignancies, including MM. Although some success has been observed, the inherent toxicities of IFNs limit their efficacy. To address this problem, we produced anti-CD138 antibody fusion proteins containing either IFNα2 or a mutant IFNα2 (IFNα2(YNS)) with the goal of targeting IFN to CD138-expressing cells, thereby achieving effective IFN concentrations at the site of the tumor in the absence of toxicity. The fusion proteins inhibited the proliferation and induced apoptosis of U266, ANBL-6, NCI-H929, and MM1-144 MM cell lines. The fusion proteins decreased the expression of IFN regulatory factor 4 (IRF4) in U266. In addition, the fusion proteins were effective against primary cells from MM patients, and treatment with fusion proteins prolonged survival in the U266 murine model of MM. These studies show that IFNα antibody fusion proteins can be effective novel therapeutics for the treatment of MM.

Type I interferons (IFNα/β) are cytokines with a broad spectrum of anti-tumor activities including anti-proliferative, pro-apoptotic, and immunostimulatory effects, and are potentially useful in the treatment of B cell malignancies and other cancers. To improve anti-tumor potency and diminish the systemic side effects of IFN, we recently developed anti-CD20-IFNα fusion proteins with in vitro and in vivo efficacy against both mouse and human lymphomas expressing CD20. Since IFNβ binds more tightly to the IFNα/β receptor (IFNAR) and has more potent anti-tumor activities, we have now constructed an anti-CD20 fusion protein with murine IFNβ (mIFNβ). Anti-CD20-mIFNβ was more potent than recombinant mIFNβ and anti-CD20-mIFNα in inhibiting the proliferation of a mouse B cell lymphoma expressing human CD20 (38C13-huCD20). Growth inhibition was accompanied by caspase-independent apoptosis and DNA fragmentation. The efficacy of anti-CD20-mIFNβ required the physical linkage of mIFNβ to anti-CD20 antibody (Ab). Importantly, anti-CD20-mIFNβ was active against tumor cells expressing low levels of IFNAR (38C13-huCD20 IFNARlo). In vivo, established 38C13-huCD20 tumors were largely insensitive to rituximab or a non-targeted mIFNβ fusion protein, yet treatment with anti-CD20-mIFNβ eradicated 83% of tumors. Anti-CD20-mIFNβ was also more potent in vivo against 38C13-huCD20 than anti-CD20-mIFNα, curing 75% versus 25% of tumors (p = 0.001). Importantly, while anti-CD20-mIFNα could not eradicate 38C13-huCD20 IFNARlo tumors, anti-CD20-mIFNβ treatment prolonged survival (p = 0.0003), and some animals remained tumor-free. Thus, Ab fusion proteins targeting mIFNβ to tumors show promise as therapeutic agents, especially for use against tumors resistant to the effects of mIFNα.

Previous studies have shown that the alternative pathway of complement activation plays an important role in protection against infection with Cryptococcus neoformans. Cryptococcus gattii does not activate the alternative pathway as well as C. neoformans in vitro. The role of complement in C. gattii infection in vivo has not been reported. In this study, we used mice deficient in complement components to investigate the role of complement in protection against a C. gattii isolate from an ongoing outbreak in northwestern North America. While factor B-deficient mice showed an enhanced rate of death, complement component C3-deficient mice died even more rapidly, indicating that the alternative pathway was not the only complement pathway contributing to protection against disease. Both C3-and factor B-deficient mice had increased fungal burdens in comparison to wild-type mice. Histopathology revealed an overwhelming fungal burden in the lungs of these complementdeficient mice, which undoubtedly prevented efficient gas exchange, causing death. Following the fate of radiolabeled organisms showed that both factor B-and C3-deficient mice were less effective than wild-type mice in clearing organisms. However, opsonization of C. gattii with complement components was not sufficient to prolong life in mice deficient in complement. Killing of C. gattii by macrophages in vitro was decreased in the presence of serum from factor B-and C3-deficient versus wild-type mice. In conclusion, we have demonstrated that complement activation is crucial for survival in C. gattii infection. Additionally, we have shown that the alternative pathway of complement activation is not the only complement pathway contributing to protection.

Although recent advances have substantially improved the management of multiple myeloma, it remains an incurable malignancy. We now demonstrate that anti-CD138 molecules genetically fused to type I interferons (IFN) synergize with the approved therapeutic bortezomib in arresting the proliferation of human multiple myeloma cell lines both in vitro and in vivo. The anti-CD138-IFNa14 fusion protein was active in inducing increased expression of signal transducer and activator of transcription 1 (STAT1) and its phosphorylation while the cell death pathway induced by bortezomib included generation of reactive oxygen species. Interferon regulatory factor 4 (IRF4), an important survival factor for myeloma cells, was down regulated following combination treatment. Induction of cell death appeared to be caspase-independent because treatment with inhibitors of caspase activation did not decrease the level of cell death. The observed caspase-independent synergistic cell death involved mitochondrial membrane depolarization, and poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, and resulted in enhanced induction of apoptosis. Importantly, using 2 different in vivo xenograft models, we found that combination therapy of anti-CD138-IFNa14 and bortezomib was able to cure animals with established tumors (7 of 8 using OCI-My5 or 8 of 8 using NCI-H929). Thus, the combination of anti-CD138-IFNa with bortezomib shows great promise as a novel therapeutic approach for the treatment of multiple myeloma, a malignancy for which there are currently no cures.