Anna Kruschinski

The Spiegelmer NOX-A12, a novel CXCL12 inhibitor, interferes with chronic lymphocytic leukemia cell motility and causes chemosensitization

The CXC chemokine ligand (CXCL12, or stromal cell-derived factor-1 as previously known) plays a critical role for homing and retention of chronic lymphocytic leukemia (CLL) cells in tissues such as the bone marrow (BM). In tissues, stromal cells constitutively secrete and present CXCL12 via cell-surface-bound glycosaminoglycans (GAGs), thereby attracting CLL cells and protecting them from cytotoxic drugs, a mechanism that may account for residual disease after conventional CLL therapy. NOX-A12, an RNA oligonucleotide in L-configuration (Spiegelmer) that binds and neutralizes CXCL12, was developed for interference with CXCL12 in the tumor microenvironment and for cell mobilization. Here, we examined effects of NOX-A12 on CLL cell migration and drug sensitivity. We found that NOX-A12 effectively inhibited CXCL12-induced chemotaxis of CLL cells. In contrast, NOX-A12 increased CLL migration underneath a confluent layer of BM stromal cells (BMSCs) due to interference with the CXCL12 gradient established by BMSCs. In particular, NOX-A12 competes with GAGs such as heparin for CXCL12 binding, leading to the release of CXCL12 from stromal cell-surface-bound GAGs, and thereby to neutralization of the chemokine. Furthermore, NOX-A12 sensitizes CLL cells toward bendamustine and fludarabine in BMSC cocultures. These data demonstrate that NOX-A12 effectively interferes with CLL cell migration and BMSC-mediated drug resistance, and establishes a rationale for clinical development of NOX-A12 in combination with conventional agents in CLL.

SDF-1 Inhibition Targets the Bone Marrow Niche for Cancer Therapy

Bone marrow (BM) metastasis remains one of the main causes of death associated with solid tumors as well as multiple myeloma (MM). Targeting the BM niche to prevent or modulate metastasis has not been successful to date. Here, we show that stromal cell-derived factor-1 (SDF-1/CXCL12) is highly expressed in active MM, as well as in BM sites of tumor metastasis and report on the discovery of the high-affinity anti-SDF-1 PEGylated mirror-image l-oligonucleotide (olaptesed-pegol). In vivo confocal imaging showed that SDF-1 levels are increased within MM cell-colonized BM areas. Using in vivo murine and xenograft mouse models, we document that in vivo SDF-1 neutralization within BM niches leads to a microenvironment that is less receptive for MM cells and reduces MM cell homing and growth, thereby inhibiting MM disease progression. Targeting of SDF-1 represents a valid strategy for preventing or disrupting colonization of the BM by MM cells.

Blockade of SDF-1 after irradiation inhibits tumor recurrences of autochthonous brain tumors in rats.

Background Tumor irradiation blocks local angiogenesis, forcing any recurrent tumor to form new vessels from circulating cells. We have previously demonstrated that the post-irradiation recurrence of human glioblastomas in the brains of nude mice can be delayed or prevented by inhibiting circulating blood vessel–forming cells by blocking the interaction of CXCR4 with its ligand stromal cell-derived factor (SDF)–1 (CXCL12). In the present study we test this strategy by directly neutralizing SDF-1 in a clinically relevant model using autochthonous brain tumors in immune competent hosts. Methods We used NOX-A12, an l-enantiomeric RNA oligonucleotide that binds and inhibits SDF-1 with high affinity. We tested the effect of this inhibitor on the response to irradiation of brain tumors in rat induced by n-ethyl-N-nitrosourea. Results Rats treated in utero with N-ethyl-N-nitrosourea began to die of brain tumors from approximately 120 days of age. We delivered a single dose of whole brain irradiation (20 Gy) on day 115 of age, began treatment with NOX-A12 immediately following irradiation, and continued with either 5 or 20 mg/kg for 4 or 8 weeks, doses and times equivalent to well-tolerated human exposures. We found a marked prolongation of rat life span that was dependent on both drug dose and duration of treatment. In addition we treated tumors only when they were visible by MRI and demonstrated complete regression of the tumors that was not achieved by irradiation alone or with the addition of temozolomide. Conclusions Inhibition of SDF-1 following tumor irradiation is a powerful way of improving tumor response of glioblastoma multiforme.

Hematopoietic stem and progenitor cell mobilization in mice and humans by a first-in-class mirror-image oligonucleotide inhibitor of CXCL12.

NOX‐A12 is a PEGylated mirror‐image oligonucleotide (a so‐called Spiegelmer) that binds to CXCL12 (stromal cell–derived factor‐1, SDF‐1) with high affinity thereby inhibiting CXCL12 signaling on both its receptors, CXCR4 and CXCR7. In animals, NOX‐A12 mobilized white blood cells (WBCs) and hematopoietic stem and progenitor cells (HSCs) into peripheral blood (PB). In healthy volunteers, single doses of NOX‐A12 had a benign safety profile and also dose‐dependently mobilized WBCs and HSCs into PB. HSC peak mobilization reached a plateau at five times the baseline level at an i.v. dose of 5.4 mg/kg. In accordance with the plasma half‐life of 38 h, the duration of the WBC and HSC mobilization was long lasting and increased dose‐dependently to more than 4 days at the highest dose (10.8 mg/kg). In conclusion, NOX‐A12 may be appropriate for therapeutic use in and beyond mobilization of HSCs, e.g., in long‐lasting mobilization and chemosensitization of hematological cancer cells.

CXCL12/SDF-1-Dependent Retinal Migration of Endogenous Bone Marrow-Derived Stem Cells Improves Visual Function after Pharmacologically Induced Retinal Degeneration

Mobilized bone marrow-derived stem cells (BMSC) have been discussed as an alternative strategy for endogenous repair. Thereby, different approaches for BMSC mobilization have been pursued. Herein, the role of a newly discovered oligonucleotide for retinal homing and regeneration capability of BMSCs was investigated in the sodium iodate (NaIO3) model of retinal degeneration. Mobilization was achieved in GFP-chimera with NOX-A12, a CXC-motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 (SDF-1)-neutralizing L-aptamer. BMSC homing was directed by intravitreal SDF-1 injection. Visual acuity was measured using the optokinetic reflex. Paraffin cross sections were stained with hematoxylin and eosin for retinal thickness measurements. Immunohistochemistry was performed to investigate the expression of cell-specific markers after mobilization. A single dose of NOX-A12 induced significant mobilization of GFP+ cells which were found in all layers within the degenerating retina. An additional intravitreal injection of SDF-1 increased migration towards the site of injury. Thereby, the number of BMSCs (Sca-1+) found in the damaged retina increased whereas a decrease of activated microglia (Iba-1+) was found. The mobilization led to significantly increased visual acuity. However, no significant changes in retinal thickness or differentiation towards retinal cell types were detected. Systemic mobilization by a single dose of NOX-A12 showed increased homing of BMSCs into the degenerated retina, which was associated with improved visual function when injection of SDF-1 was additionally performed. The redistribution of the cells to the site of injury combined with their observed beneficial effects support the endogenous therapeutic strategy for retinal repair.

Abstract PR14: In vivo targeting of stromal-derived factor-1 as a strategy to prevent myeloma cell dissemination to distant bone marrow niches

Background. Multiple myeloma (MM) patients present with multiple lytic lesions at diagnosis, indicating the presence of continuous dissemination of MM cells from the primary site of tumor development to multiple distant bone marrow (BM) niches. We hypothesized that stromal-derived factor-1 (SDF1) may represent a target for preventing transition from MGUS (micrometastatic-stage) to active-MM (macrometastatic-stage). We therefore evaluated SDF1 expression in the BM of patients with MGUS, MM, compared to healthy individuals; and tested NOX-A12, a high affinity L-oligonucleotide binder to SDF1, looking at its ability to modulate MM cell tumor growth and homing to the BM in vivo and in vitro. Methods. SDF1 levels were evaluated by immunohistochemistry on BM specimens of patients with MGUS, active-MM, or healthy individuals; and confirmed by ELISA, using conditioned-medium of BM-mesenchymal stromal cells (BM-MSCs) from MGUS, active-MM and healthy individuals. BM metastatic lesions from primary epithelial tumors were also evaluated. Co-localization of SDF1 with MM cell (MM.1S-GFP + )-enriched BM niches was evaluated using in vivo confocal microscopy. Effect of NOX-A12 on modulating MM cell dissemination was tested in vivo, by using in vivo MM metastasis model. In vivo homing and in vivo tumor growth of MM cells (MM.1S-GFP + /luc + ) was assessed by using in vivo confocal microscopy and bioluminescence, in mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12+bortezomib. Effects of drug combination on dissemination of MM cells to distant BM niches was evaluated ex vivo by immunofluorescence on explanted femurs. DNA synthesis and adhesion of MM cells in the context of NOX-A12 treated primary MM BM-MSCs in presence or absence of bortezomib were tested by thymidine uptake and adhesion in vitro assay, respectively. Synergism was calculated by using CalcuSyn software. Results. Patients with active-MM present with higher BM SDF1 expression vs MGUS patients and healthy individuals. Similarly, BM presenting with metastasis from epithelial primary tumors had higher SDF1 levels compared to healthy subjects, thus suggesting the importance of SDF1 in favoring tumor cell metastasis to BM niches. SDF1 co-localized at BM level with MM tumor cells in vivo. In vitro, NOX-A12 induced a dose-dependent de-adhesion of MM cells from the BMSCs supported by inhibition of BM-MSC-mediated phosphorylation of ERK1/2 and cofilin. Importantly, NOX-A12 induced MM cell mobilization from the BM to the peripheral blood (PB) as shown ex vivo by reduction of MM cells in the BM and increased number of MM cells within the PB compared to control mice (P Conclusion. SDF-1 represents a valid target for inhibiting MM cell dissemination to distant BM niches, thus providing the evidence for using the SDF1 inhibiting spiegelmer NOX-A12 to target MM cells at the stage of micrometastasis (MGUS), thus preventing development of macrometastatic MM. This abstract is also presented as Poster B80. Citation Format: Aldo M. Roccaro, Antonio Sacco, Michele Moschetta, Patricia Maiso, Yuji Mishima, Yosra Aljawai, Fabio Facchetti, Anna Kruschinski, Giuseppe Rossi, Irene M. Ghobrial. In vivo targeting of stromal-derived factor-1 as a strategy to prevent myeloma cell dissemination to distant bone marrow niches. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR14. doi:10.1158/1538-7445.CHTME14-PR14

Abstract 385: Inhibition of recurrences of experimental brain tumors after irradiation by blocking the activity of SDF-1 (CXCL12) using the Spiegelmer® NOX-A12.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC With current treatment of glioblastomas 85% of the tumors recur within the field of high dose radiation. This tumor recurrence requires formation of new blood vessels. We have developed a new therapeutic paradigm based on the dual origin of tumor blood vessels: Angiogenesis - the sprouting of endothelial cells from nearby blood vessels, and vasculogenesis - the formation of blood vessels from circulating cells. Because tumor irradiation abrogates local angiogenesis the tumor must rely on the vasculogenesis pathway for regrowth after irradiation. We have previously demonstrated that the post-irradiation recurrence of human glioblastomas implanted intracranially in the mouse brain can be delayed or prevented by inhibiting circulating blood vessel forming cells by blocking the interaction of the chemokine receptor CXCR4 with its ligand SDF-1 (CXCL12)1. However, SDF-1 has a second receptor, CXCR7, which has been implicated in endothelial cell migration, is present on tumor vasculature, and is potentially also able to activate vasculogenesis. To block both receptors we have therefore used NOX-A12, an L-enantiomeric RNA oligonucleotide (Spiegelmer®), which inhibits SDF-1 with subnanomolar affinity. We have tested the effect of this inhibitor on the response to irradiation of the U251 human GBM in nude mice and on ENU-induced brain tumors in the Sprague-Dawley rat. In the mouse model we found that inhibition of SDF-1 post-irradiation was highly effective in enhancing tumor response to a single dose of 20 Gy whole brain irradiation. In the rat model the pups from ENU-treated pregnant mothers begin to die of brain tumors from approximately 120 days of age. We performed two studies with this model: In the first we delivered a single dose of whole brain irradiation (20 Gy) on day 115 of age and began treatment with NOX-A12 immediately following irradiation and continued every 2 days with either 5 or 20 mg/kg injected subcutaneously for either 4 or 8 weeks. These doses and times were chosen as equivalent to human doses and times that based on existing data have been found to be safe and well tolerated in human volunteers and which are effective in inhibiting the action of SDF-1. We found a marked prolongation of life of the rats that was dependent both on drug dose and length of treatment. In the second study we measured the size of the tumors post-irradiation using MRI and again found a pronounced enhancement of the efficacy of irradiation that was superior to the addition of temozolomide at doses equivalent to those used with concomitant radiotherapy in treating GBM. We believe that these encouraging data justify a human trial in first line glioblastoma patients. 1. Kioi M, Vogel H, Schultz G, Hoffman RM, Harsh GR, Brown JM: Inhibition of vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation in mice. J Clin Invest 120:694-705, 2010 Citation Format: Shie-Chau Liu, Reem Alomran, Jason Stafford, Milton Merchant, Taichang Jang, Stefan Zollner, Anna Kruschinski, Laurence Recht, J. Martin Brown. Inhibition of recurrences of experimental brain tumors after irradiation by blocking the activity of SDF-1 (CXCL12) using the Spiegelmer® NOX-A12. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 385. doi:10.1158/1538-7445.AM2013-385

Abstract 4382: Inhibition of SDF-1 (CXCL12) using the Spiegelmer NOX-A12 markedly delays the recurrence of ENU-induced rat brain tumors following irradiation

We have previously shown that the post-irradiation recurrence of human glioblastomas implanted intracranially in the mouse brain can be delayed or prevented by inhibiting the interaction of the chemokine receptor CXCR4 with its ligand SDF-1 (CXCL12)1. This effect is due to inhibition of the post-irradiation recovery of the tumor vasculature from circulating cells, a process known as vasculogenesis. However, SDF-1 has a second receptor, CXCR7, which has been implicated in endothelial cell migration2, is present on tumor vasculature2, and is potentially also able to activate vasculogenesis. Therefore we investigated the efficacy of the SDF-1 inhibitor NOX-A12, an L-enantiomeric RNA oligonucleotide (Spiegelmer), on brain tumor recurrences after irradiation. NOX-A12 inhibits SDF-1 with subnanomolar affinity and should therefore inhibit SDF-1 mediated activation of both receptors, CXCR4 and CXCR7. In this study we used ENU-induced brain tumors in the Sprague-Dawley rat, a model that has proved extremely resistant to anticancer therapy in prior studies by a variety of investigators. Pregnant rats were injected with the carcinogen ethylnitrosourea (50mg/kg) on day 17 of gestation. In this model the pups appear normal at birth but begin to die of brain tumors from approximately 120 days of age. We delivered a single dose of whole brain irradiation (20 Gy) on day 115 of age and began treatment with NOX-A12 immediately following irradiation and continued every 2 days with either 5 or 20 mg/kg injected subcutaneously for either 4 or 8 weeks. These doses and times were chosen as equivalent to human doses and times that based on existing data have been found to be safe and well tolerated in human volunteers and which are effective in inhibiting the action of SDF-1. We found that neither 20 Gy nor NOX-A12 alone prolonged the lifespan of the tumor-bearing rats. However, the addition of NOX-A12 to 20 Gy prolonged the lifespan of the rats particularly at the highest dose and for the longer treatment period of 8 weeks (median lifespans of 20 Gy alone and 20 Gy + 5 and 20 mg/kg of NOX-A12 were 196, 291 and 349 days respectively with p values for NOX-A12 treated rats Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4382. doi:1538-7445.AM2012-4382