William H. Towns

A Mouse Model for the Carney Complex Tumor Syndrome Develops Neoplasia in Cyclic AMP–Responsive Tissues

Carney complex is an autosomal dominant neoplasia syndrome characterized by spotty skin pigmentation, myxomatosis, endocrine tumors, and schwannomas. This condition may be caused by inactivating mutations in PRKAR1A, the gene encoding the type 1A regulatory subunit of protein kinase A. To better understand the mechanism by which PRKAR1A mutations cause disease, we have developed conventional and conditional null alleles for Prkar1a in the mouse. Prkar1a(+/-) mice developed nonpigmented schwannomas and fibro-osseous bone lesions beginning at approximately 6 months of age. Although genotype-specific cardiac and adrenal lesions were not seen, benign and malignant thyroid neoplasias were observed in older mice. This spectrum of tumors overlaps that seen in Carney complex patients, confirming the validity of this mouse model. Genetic analysis indicated that allelic loss occurred in a subset of tumor cells, suggesting that complete loss of Prkar1a plays a key role in tumorigenesis. Similarly, tissue-specific ablation of Prkar1a from a subset of facial neural crest cells caused the formation of schwannomas with divergent differentiation. These observations confirm the identity of PRKAR1A as a tumor suppressor gene with specific importance to cyclic AMP-responsive tissues and suggest that these mice may be valuable tools not only for understanding endocrine tumorigenesis but also for understanding inherited predispositions for schwannoma formation.

17-DMAG targets the nuclear factor-κB family of proteins to induce apoptosis in chronic lymphocytic leukemia: clinical implications of HSP90 inhibition

The HSP90 client chaperone interaction stabilizes several important enzymes and antiapoptotic proteins, and pharmacologic inhibition of HSP90 results in rapid client protein degradation. Therefore, HSP90 inhibition is an attractive therapeutic approach when this protein is active, a phenotype commonly observed in transformed but not normal cells. However, preclinical studies with HSP90 inhibitors such as 17-AAG demonstrated depletion of only a subset of client proteins and very modest tumor cytotoxicity in chronic lymphocytic leukemia (CLL) cells. Herein, we describe another HSP90 inhibitor, 17-DMAG, which is cytotoxic to CLL but not normal lymphocytes. Treatment with 17-DMAG leads to depletion of the HSP90 client protein IKK, resulting in diminished NF-kappaB p50/p65 DNA binding, decreased NF-kappaB target gene transcription, and caspase-dependent apoptosis. Furthermore, treatment with 17-DMAG significantly decreased the white blood cell count and prolonged the survival in a TCL1-SCID transplant mouse model. The ability of 17-DMAG to function as an NF-kappaB inhibitor is of great interest clinically, as few currently available CLL drugs target this transcription factor. Therefore, the effect of 17-DMAG on NF-kappaB signaling pathways represents a novel therapy warranting further clinical pursuit in this and other B-cell lymphoproliferative disorders.

Bruton’s tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukemia (CLL) is characterized by constitutive activation of the B-cell receptor (BCR) signaling pathway, but variable responsiveness of the BCR to antigen ligation. Brutons tyrosine kinase (BTK) shows constitutive activity in CLL and is the target of irreversible inhibition by ibrutinib, an orally bioavailable kinase inhibitor that has shown outstanding activity in CLL. Early clinical results in CLL with other reversible and irreversible BTK inhibitors have been less promising, however, raising the question of whether BTK kinase activity is an important target of ibrutinib and also in CLL. To determine the role of BTK in CLL, we used patient samples and the Eμ-TCL1 (TCL1) transgenic mouse model of CLL, which results in spontaneous leukemia development. Inhibition of BTK in primary human CLL cells by small interfering RNA promotes apoptosis. Inhibition of BTK kinase activity through either targeted genetic inactivation or ibrutinib in the TCL1 mouse significantly delays the development of CLL, demonstrating that BTK is a critical kinase for CLL development and expansion and thus an important target of ibrutinib. Collectively, our data confirm the importance of kinase-functional BTK in CLL.

Ibrutinib treatment ameliorates murine chronic graft-versus-host disease

Chronic graft-versus-host disease (cGVHD) is a life-threatening impediment to allogeneic hematopoietic stem cell transplantation, and current therapies do not completely prevent and/or treat cGVHD. CD4+ T cells and B cells mediate cGVHD; therefore, targeting these populations may inhibit cGVHD pathogenesis. Ibrutinib is an FDA-approved irreversible inhibitor of Brutons tyrosine kinase (BTK) and IL-2 inducible T cell kinase (ITK) that targets Th2 cells and B cells and produces durable remissions in B cell malignancies with minimal toxicity. Here, we evaluated whether ibrutinib could reverse established cGVHD in 2 complementary murine models, a model interrogating T cell-driven sclerodermatous cGVHD and an alloantibody-driven multiorgan system cGVHD model that induces bronchiolar obliterans (BO). In the T cell-mediated sclerodermatous cGVHD model, ibrutinib treatment delayed progression, improved survival, and ameliorated clinical and pathological manifestations. In the alloantibody-driven cGVHD model, ibrutinib treatment restored pulmonary function and reduced germinal center reactions and tissue immunoglobulin deposition. Animals lacking BTK and ITK did not develop cGVHD, indicating that these molecules are critical to cGVHD development. Furthermore, ibrutinib treatment reduced activation of T and B cells from patients with active cGVHD. Our data demonstrate that B cells and T cells drive cGVHD and suggest that ibrutinib has potential as a therapeutic agent, warranting consideration for cGVHD clinical trials.

Heart-Specific Ablation of Prkar1a Causes Failure of Heart Development and Myxomagenesis

Background— Protein kinase A signaling has long been known to play an important role in cardiac function. Dysregulation of the protein kinase A system, caused by mutation of the protein kinase A regulatory subunit gene PRKAR1A, causes the inherited tumor syndrome Carney complex, which includes cardiac myxomas as one of its cardinal features. Mouse models of this genetic defect have been unsatisfactory because homozygote null animals die early in development and heterozygotes do not exhibit a cardiac phenotype. Methods and Results— To study the cardiac-specific effects resulting from complete loss of Prkar1a, we used cre-lox technology to generate mice lacking this protein specifically in cardiomyocytes. Conditional knockout mice died at day 11.5 to 12.5 of embryogenesis with thin-walled, dilated hearts. These hearts showed elevated protein kinase A activity and decreased cardiomyocyte proliferation before demise. Analysis of the expression of transcription factors required for cardiogenesis revealed downregulation of key cardiac transcription factors such as the serum response factor, Gata4, and Nkx2–5. Although heart wall thickness was reduced overall, specific areas exhibited morphological changes consistent with myxomatous degeneration in the walls of knockout hearts. Conclusions— Loss of Prkar1a from the heart causes a failure of proper myocardial development with subsequent cardiac failure and embryonic demise. These changes appear to be due to suppression of cardiac-specific transcription by increased protein kinase A activity. These biochemical changes lead to myxoma-like changes, indicating that these mice may be a good model with which to study the formation of these tumors.

FTY720 increases CD74 expression and sensitizes mantle cell lymphoma cells to milatuzumab-mediated cell death

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a short median survival despite multimodal therapy. FTY720, an immunosuppressive drug approved for the treatment of multiple sclerosis, promotes MCL cell death concurrent with down-modulation of phospho-Akt and cyclin D1 and subsequent cell-cycle arrest. However, the mechanism of FTY720-mediated MCL cell death remains to be fully clarified. In the present study, we show features of autophagy blockage by FTY720 treatment, including accumulation of autolysosomes and increased LC3-II and p62 levels. We also show that FTY720-induced cell death is mediated by lysosomal membrane permeabilization with subsequent translocation of lysosomal hydrolases to the cytosol. FTY720-mediated disruption of the autophagic-lysosomal pathway led to increased levels of CD74, a potential therapeutic target in MCL that is degraded in the lysosomal compartment. This finding provided rationale for examining combination therapy with FTY720 and milatuzumab, an anti-CD74 mAb. Treatment of MCL cell lines and primary tumor cells with FTY720 and milatuzumab resulted in statistically significant enhanced cell death, which was synergistic in blastic variant MCL cell lines. Significant in vivo therapeutic activity of combination treatment was also demonstrated in a preclinical, in vivo model of MCL. These findings support clinical evaluation of this combination in patients with MCL.

Milatuzumab-Conjugated Liposomes as Targeted Dexamethasone Carriers for Therapeutic Delivery in CD74+ B-cell Malignancies

Purpose: Corticosteroids are widely used for the treatment of B-cell malignancies, including non–Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and acute lymphoblastic leukemia; however, this class of drug is associated with undesirable off-target effects. Herein, we developed novel milatuzumab-conjugated liposomes as a targeted dexamethasone carrier for therapeutic delivery in CD74+ B-cell malignancies and explored its effect against the disease. Experimental Design: The targeting efficiency of milatuzumab-targeted liposomes to CD74+ cells was evaluated in vitro. The effect of CD74-targeted liposomal dexamethasone was compared with free dexamethasone in primary CLL cells and cell lines in vitro. The therapeutic efficacy of CD74-targeted liposomal dexamethasone was evaluated in a Raji-severe combined immunodeficient (SCID) xenograft model in vivo. Results: Milatuzumab-targeted liposomes promoted selective incorporation of carrier molecules into transformed CD74-positive B cells as compared with CD74-negative T-cells. The CD74-dexamethasone–targeted liposomes (CD74-IL-DEX) promoted and increased killing in CD74-positive tumor cells and primary CLL cells. Furthermore, the targeted drug liposomes showed enhanced therapeutic efficacy against a CD74-positive B-cell model as compared with free, or non-targeted, liposomal dexamethasone in SCID mice engrafted with Raji cells in vivo. Conclusions: These studies provide evidence and support for a potential use of CD74-targeted liposomal dexamethasone as a new therapy for B-cell malignancies. Clin Cancer Res; 19(2); 347–56. ©2012 AACR.

Dual Targeting of the Cyclin/Rb/E2F and Mitochondrial Pathways in Mantle Cell Lymphoma with the Translation Inhibitor Silvestrol

Purpose: During cell-cycle progression, D-cyclins activate cyclin-dependent kinases (CDKs) 4/6 to inactivate Rb, permitting E2F1-mediated S-phase gene transcription. This critical pathway is typically deregulated in cancer, and novel inhibitory strategies would be effective in a variety of tumors. The protein synthesis inhibitor silvestrol has potent activity in B-cell leukemias via the mitochondrial pathway of apoptosis, and also reduces cyclin D1 expression in breast cancer and lymphoma cell lines. We hypothesized that this dual activity of silvestrol would make it especially effective in malignancies driven by aberrant cyclin D1 expression. Experimental Design: Mantle cell lymphoma (MCL), characterized by elevated cyclin D1, was used as a model to test this approach. The cyclin D/Rb/E2F1 pathway was investigated in vitro using MCL cell lines and primary tumor cells. Silvestrol was also evaluated in vivo using an aggressive model of MCL. Results: Silvestrol showed low nanomolar potency both in MCL cell lines and primary MCL tumor cells. D-cyclins were depleted with just 10 nmol/L silvestrol at 16 hours, with subsequent reductions of phosphorylated Rb, E2F1 protein, and E2F1 target transcription. As showed in other leukemias, silvestrol caused Mcl-1 depletion followed by mitochondrial depolarization and caspase-dependent apoptosis, effects not related to inhibition of CDK4/6. Silvestrol significantly (P < 0.0001) prolonged survival in a MCL xenograft model without detectable toxicity. Conclusions: These data indicate that silvestrol effectively targets the cyclin/CDK/Rb pathway, and additionally induces cytotoxicity via intrinsic apoptosis. This dual activity may be an effective therapeutic strategy in MCL and other malignancies. Clin Cancer Res; 18(17); 4600–11. ©2012 AACR.

Characterization of a New Chronic Lymphocytic Leukemia Cell Line for Mechanistic In Vitro and In Vivo Studies Relevant to Disease

Studies of chronic lymphocytic leukemia (CLL) have yielded substantial progress, however a lack of immortalized cell lines representative of the primary disease has hampered a full understanding of disease pathogenesis and development of new treatments. Here we describe a novel CLL cell line (OSU-CLL) generated by EBV transformation, which displays a similar cytogenetic and immunophenotype observed in the patient’s CLL (CD5 positive with trisomy 12 and 19). A companion cell line was also generated from the same patient (OSU-NB). This cell line lacked typical CLL characteristics, and is likely derived from the patient’s normal B cells. In vitro migration assays demonstrated that OSU-CLL exhibits migratory properties similar to primary CLL cells whereas OSU-NB has significantly reduced ability to migrate spontaneously or towards chemokine. Microarray analysis demonstrated distinct gene expression patterns in the two cell lines, including genes on chromosomes 12 and 19, which is consistent with the cytogenetic profile in this cell line. Finally, OSU-CLL was readily transplantable into NOG mice, producing uniform engraftment by three weeks with leukemic cells detectable in the peripheral blood spleen and bone marrow. These studies describe a new CLL cell line that extends currently available models to study gene function in this disease.

Abstract 2591: Ibrutinib can reverse established chronic graft-versus-host disease, which is dependent upon IL-2 inducible T-cell kinase (ITK) and Bruton's tyrosine kinase (BTK)-driven lymphocyte activation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Chronic graft versus host disease (cGVHD) is a life threatening impediment to the otherwise curative potential of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Intensive study has unveiled the lymphoid subsets that drive multifaceted autoimmune and fibrotic cascades which disrupt healthy tissues. Clinical and in-vivo data implicate B-cells and certain subsets of CD4 helper T-cells in the development and progression of cGVHD. These specific immune subsets are unique in their singular dependence upon the TEC-family kinases BTK or ITK for receptor driven activation. Ibrutinib is an irreversible inhibitor of BTK and ITK that has produced durable remissions in B-cell malignancies with minimal toxicity. We sought to examine ibrutinibs potential to attenuate B-cell and CD4 T-cell driven anti-host immunity in the setting of cGVHD. We utilized two complementary murine models independently focused on interrogating sclerodermatous T-cell driven cGVHD (LPJ→C57BL/6) or alloantibody driven bronchiolar obliterans (BO) and multi-organ system disease without skin involvement (C57BL/6→B10.BR). We examined therapeutic administration of 25mg/kg/day ibrutinib initiated when mice first develop signs of cGVHD (25-28 days after allo-HSCT). In the minor-MHC mismatch LPJ→C57BL/6 model, ibrutinib significantly extended median time to cGVHD progression by 14 days and 33% (6 of 18) of ibrutinib treated mice remained progression free as compared to 12% (2 of 18) of mice receiving vehicle and 10% (1 of 11) of mice receiving cyclosporine 10mg/kg/day (p<0.02). Overall survival was also highest in the ibrutinib treatment cohort. Mice receiving ibrutinib showed restoration of external cGVHD criterion including body weight, posture, skin lesions, hair loss, and mobility. There was significant ablation of lymphohistiocytic T- and B-cell pulmonary and renal infiltration in ibrutinib treated animals. Data from the C57BL/6→B10.BR model confirmed the efficacy of ibrutinib in significantly restoring pulmonary compliance, elastance, and resistance to levels indistinguishable from that of a healthy animal and significantly better than cGVHD vehicle treated controls (n=12/group)(p<0.01). These results were associated with significantly decreased alloantibody deposition and decreased fibrosis in the lungs of ibrutinib treated mice. Germinal center (GC) reactions, required for multi-organ system pathology in this model, were also significantly reduced within the spleens of mice receiving therapeutic ibrutinib (p<0.05). Using in-vivo genetic ablation models, we demonstrate the critical nature of BTK and ITK respectively in the development of cGVHD pathognomonic BO. Our data support ibrutinib as a promising therapeutic direction for the treatment of cGVHD by virtue of its ability to suppress BTK and ITK signaling. Citation Format: Jason A. Dubovsky, Ryan Flynn, Jing Du, Bonnie K. Harrington, Yiming Zhong, Carrie Yang, William Towns, Amy Lehman, Amy Johnson, Steven Devine, Samantha Jaglowski, Jonathan S. Serody, William J. Murphy, David H. Munn, Leo Luznik, Geoffrey Hill, Kelli K.P. MacDonald, Ivan Maillard, John Koreth, Corey Cutler, Robert J. Soiffer, Joseph H. Antin, Jerome Ritz, Angela Panoskaltsis-Mortari, John C. Byrd, Bruce R. Blazar. Ibrutinib can reverse established chronic graft-versus-host disease, which is dependent upon IL-2 inducible T-cell kinase (ITK) and Brutons tyrosine kinase (BTK)-driven lymphocyte activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2591. doi:10.1158/1538-7445.AM2014-2591