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Supplementary Figure Legends

Supplementary Table 4: Combination Index (CI) Values. CI values calculated using the Chou and Talalay method from SRB proliferation data for varying concentrations of Nutlin and MLN8237 in combination. CI < 1 indicates synergy; CI = 1 additivity; CI > 1 antagonism

Supplementary Figure 2. Quantification of total cell counts and average cellular size at Day 15 as depicted in Fig. 4A.

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Supplementary Table 3: Quantification of total cell counts and average cellular size under conditions depicted in Fig. 4A.

Supplementary Figure 4. Effect of MLN8237 on Cleaved Caspase-3 in TNBC xenografts in vivo.

Supplementary Figure 1. Treatment with MLN8237 induces markers of cellular senescence in two additional CAL-51 p53 and p73 shRNA knockdown clones (p53-12, p73-26).

Supplementary Table 2: Sensitivity of CAL-51 KD clones to MLN8237 in vitro.

The dual pathway inhibitor rigosertib inhibits phosphoinositide 3-kinase (PI3K) pathway activation as well as polo-like kinase 1 (PLK1) activity across a broad spectrum of cancer cell lines. The importance of PIK3CA alterations in squamous cell carcinoma of the head and neck (HNSCC) has raised interest in exploring agents targeting PI3K, the product of PIK3CA. The genetic and molecular basis of rigosertib treatment response was investigated in a panel of 16 HNSCC cell lines, and direct patient tumor xenografts from eight patients with HNSCC [four HPV-serotype16 (HPV16)–positive]. HNSCC cell lines and xenografts were characterized by pathway enrichment gene expression analysis, exon sequencing, gene copy number, Western blotting, and immunohistochemistry (IHC). Rigosertib had potent antiproliferative effects on 11 of 16 HPV− HNSCC cell lines. Treatment sensitivity was confirmed in two cell lines using an orthotopic in vivo xenograft model. Growth reduction after rigosertib treatment was observed in three of eight HNSCC direct patient tumor lines. The responsive tumor lines carried a combination of a PI3KCA-activating event (amplification or mutation) and a p53-inactivating event (either HPV16- or mutation-mediated TP53 inactivation). In this study, we evaluated the in vitro and in vivo efficacy of rigosertib in both HPV+ and HPV− HNSCCs, focusing on inhibition of the PI3K pathway. Although consistent inhibition of the PI3K pathway was not evident in HNSCC, we identified a combination of PI3K/TP53 events necessary, but not sufficient, for rigosertib sensitivity. Mol Cancer Ther; 12(10); 1994–2005. ©2013 AACR.

Table 1: Cell lines, fusions, tyrosine kinase inhibitors and targets. Table 2: IC50 values for Figure 1C and relative fold change.

Oncogenic kinase fusions of ALK, ROS1, RET, and NTRK1 act as drivers in human lung and other cancers. Residual tumor burden following treatment of ALK or ROS1+ lung cancer patients with oncogene-targeted therapy ultimately enables the emergence of drug-resistant clones, limiting the long-term effectiveness of these therapies. To determine the signaling mechanisms underlying incomplete tumor cell killing in oncogene-addicted cancer cells, we investigated the role of EGFR signaling in drug-naïve cancer cells harboring these oncogene fusions. We defined three distinct roles for EGFR in the response to oncogene-specific therapies. First, EGF-mediated activation of EGFR blunted fusion kinase inhibitor binding and restored fusion kinase signaling complexes. Second, fusion kinase inhibition shifted adaptor protein binding from the fusion oncoprotein to EGFR. Third, EGFR enabled bypass signaling to critical downstream pathways such as MAPK. While evidence of EGFR-mediated bypass signaling has been reported after ALK and ROS1 blockade, our results extended this effect to RET and NTRK1 blockade and uncovered the other additional mechanisms in gene fusion–positive lung cancer cells, mouse models, and human clinical specimens before the onset of acquired drug resistance. Collectively, our findings show how EGFR signaling can provide a critical adaptive survival mechanism that allows cancer cells to evade oncogene-specific inhibitors, providing a rationale to cotarget EGFR to reduce the risks of developing drug resistance. Cancer Res; 77(13); 3551–63. ©2017 AACR.

Fig. S1: EGFR inhibition or stimulation affects normal and catalytically inactive fusion oncogene signaling. Fig. S2: EGFR knockdown reduces fusion kinase cancer cell proliferation. Fig. S3: Conserved re-phosphorylated activation loop tyrosines enabled signaling PLA design and representative image analyses of fusion kinase and EGFR signaling PLAs. Fig. S4: Optimization of the EGFR-GRB2 PLA and fusion kinase PLA antibody controls. Fig. S5: The GRB2 adaptor does not switch from ROS1 to MET with FKI treatment, but MET can signal through GRB2. FIg. S6: GRB2 and SHC1 signaling rewire in a RET+ cell line treated with a fusion kinase inhibitor (FKI). Fig. S7: TRK, RET, ROS1 and ALK fusion kinases interact specifically with EGFR. Fig. S8: EGFR is highly expressed, phosphorylated, and signaling in vivo in an NTRK1+ patient-derived xenograft (PDX) model. Fig. S9: Fusion kinase, and EGFR signaling complexes are present in RET, and ALK, fusion resistant patient samples.

PDF file - 1255K, Confirmation of GLI1 modulation by EGFR activation (S1); Epithelial gene expression profile and morphology predicts erlotinib sensitivity in HNSCC cell lines (S2); Zeb1 transcription factor is key driver of EGF induced EMT (S3); EGFR activation induces EMT in HNSCC cell lines characterized by expression of Vimentin (S4); Inter-pathway crosstalk as well as EGF induced EMT are blunted in erlotinib resistant cells (S5); Silencing of HhP transcription factor GLI1 increases EGFR driven EMT in Tu-167 cells (S6); MEK/ERK signaling is required for EMT-like state while both MEK/ERK and PI3K/AKT are required for invasion through Matrigel (S7); Proliferative inhibition of erltotinib (1μM and 2μM) plus IPI-926 (1μM) or SHH (250ng/ml) (S8); Cetuximab and IPI-926 modulate EGFR, HhP and EMT gene expression in vivo (S9).

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Supplemental Methods. Description of additional methods and procedures used in the study.

PDF file - 29K, EGFR dependent EMT quantified as percent Vimentin-positive cells in culture (S1); Combination therapy delays or blocks tumor re-growth in sensitive xenografts (S2).

Supplemental Tables S1-S2. Hirsch Score was calculated from tumors and their respective stroma from Figure 5 (S1); All potential flank implanted stromal:tumor and tumor:tumor combinations were measured bi-weekly to chart growth kinetics (S2).

Supplemental Figures S1-S6. Comparison of HN11 and TU167 by RNA-Seq Expression (S1); Suppression of Radiation-induced GLI1 using shRNA Model (S2); Cyst Formation Following Radiation Treatment in Orthotopic TU167-implanted Tumors (S3); Radiation-induced Gli1 Expression and the Effect of Rapamycin Inhibition in HN11 Cells (S4); Effect of siRNA inhibition on Head Neck Cancer Cells and Gli1 Nuclear Translocation (S5); Schema and Histology Detailing Radiation-induced Tumor Repopulation (S6).

Supplemental Table and Figure Legends. Legends for Supplemental Figures S1-S6 and Supplemental Tables S1-S2.

Oncogenic kinase fusions of ALK, ROS1, RET, and NTRK1 act as drivers in human lung and other cancers. Residual tumor burden following treatment of ALK or ROS1+ lung cancer patients with oncogene-targeted therapy ultimately enables the emergence of drug-resistant clones, limiting the long-term effectiveness of these therapies. To determine the signaling mechanisms underlying incomplete tumor cell killing in oncogene-addicted cancer cells, we investigated the role of EGFR signaling in drug-naïve cancer cells harboring these oncogene fusions. We defined three distinct roles for EGFR in the response to oncogene-specific therapies. First, EGF-mediated activation of EGFR blunted fusion kinase inhibitor binding and restored fusion kinase signaling complexes. Second, fusion kinase inhibition shifted adaptor protein binding from the fusion oncoprotein to EGFR. Third, EGFR enabled bypass signaling to critical downstream pathways such as MAPK. While evidence of EGFR-mediated bypass signaling has been reported after ALK and ROS1 blockade, our results extended this effect to RET and NTRK1 blockade and uncovered the other additional mechanisms in gene fusion–positive lung cancer cells, mouse models, and human clinical specimens before the onset of acquired drug resistance. Collectively, our findings show how EGFR signaling can provide a critical adaptive survival mechanism that allows cancer cells to evade oncogene-specific inhibitors, providing a rationale to cotarget EGFR to reduce the risks of developing drug resistance. Cancer Res; 77(13); 3551–63. ©2017 AACR.

Triple-negative breast cancer (TNBC) is an aggressive disease with a poor prognosis. Advances in the treatment of TNBC have been hampered by the lack of novel effective targeted therapies. The primary goal of this study was to evaluate the efficacy of targeting Aurora kinase A (AurA), a key regulator of mitosis, in TNBC models. A secondary objective was to determine the role of the p53 family of transcriptional regulators, commonly mutated in TNBC, in determining the phenotypic response to the AurA inhibitor alisertib (MLN8237). Alisertib exhibited potent antiproliferative and proapoptotic activity in a subset of TNBC models. The induction of apoptosis in response to alisertib exposure was dependent on p53 and p73 activity. In the absence of functional p53 or p73, there was a shift in the phenotypic response following alisertib exposure from apoptosis to cellular senescence. In addition, senescence was observed in patient-derived tumor xenografts with acquired resistance to alisertib treatment. AurA inhibitors are a promising class of novel therapeutics in TNBC. The role of p53 and p73 in mediating the phenotypic response to antimitotic agents in TNBC may be harnessed to develop an effective biomarker selection strategy in this difficult to target disease. Mol Cancer Ther; 14(5); 1117–29. ©2015 AACR.

The limitations of cancer cell lines have led to the development of direct patient derived xenograft (PDX) models. However, the interplay between the implanted human cancer cells and recruited mouse stromal and immune cells alters the tumor microenvironment and limits the value of these models. To overcome these constraints, we have developed a technique to expand human hematopoietic stem and progenitor cells (HSPCs) and use them to reconstitute the radiation-depleted bone marrow of a NOD/SCID/IL2rg−/− (NSG) mouse on which a patient’s tumor is then transplanted (XactMice). The human HSPCs produce immune cells that home into the tumor and help replicate its natural microenvironment. Despite previous passage on nude mice, the expression of epithelial, stromal, and immune genes in XactMice tumors aligns more closely to that of the patient tumor than to those grown in non-humanized mice – an effect partially facilitated by human cytokines expressed by both the HSPC progeny and the tumor cells. The human immune and stromal cells produced in the XactMice can help recapitulate the microenvironment of an implanted xenograft, reverse the initial genetic drift seen after passage on non-humanized mice, and provide a more accurate tumor model to guide patient treatment.

The EGF receptor (EGFR)-directed monoclonal antibody cetuximab is the only targeted therapy approved for the treatment of squamous cell carcinoma of the head and neck (HNSCC) but is only effective in a minority of patients. Epithelial-to-mesenchymal transition (EMT) has been implicated as a drug resistance mechanism in multiple cancers, and the EGFR and Hedgehog pathways (HhP) are relevant to this process, but the interplay between the two pathways has not been defined in HNSCC. Here, we show that HNSCC cells that were naturally sensitive to EGFR inhibition over time developed increased expression of the HhP transcription factor GLI1 as they became resistant after long-term EGFR inhibitor exposure. This robustly correlated with an increase in vimentin expression. Conversely, the HhP negatively regulated an EGFR-dependent, EMT-like state in HNSCC cells, and pharmacologic or genetic inhibition of HhP signaling pushed cells further into an EGFR-dependent phenotype, increasing expression of ZEB1 and VIM. In vivo treatment with cetuximab resulted in tumor shrinkage in four of six HNSCC patient-derived xenografts; however, they eventually regrew. Cetuximab in combination with the HhP inhibitor IPI-926 eliminated tumors in two cases and significantly delayed regrowth in the other two cases. Expression of EMT genes TWIST and ZEB2 was increased in sensitive xenografts, suggesting a possible resistant mesenchymal population. In summary, we report that EGFR-dependent HNSCC cells can undergo both EGFR-dependent and -independent EMT and HhP signaling is a regulator in both processes. Cetuximab plus IPI-926 forces tumor cells into an EGFR-dependent state, delaying or completely blocking tumor recurrence. Cancer Res; 73(11); 3381–92. ©2013 AACR.

Targeted therapy development in head and neck squamous cell carcinoma (HNSCC) is challenging given the rarity of activating mutations. Additionally, HNSCC incidence is increasing related to human papillomavirus (HPV). We sought to develop an in vivo model derived from patients reflecting the evolving HNSCC epidemiologic landscape, and use it to identify new therapies. Primary and relapsed tumors from HNSCC patients, both HPV+ and HPV-, were implanted on mice, giving rise to 25 strains. Resulting xenografts were characterized by detecting key mutations, measuring protein expression by IHC and gene expression/pathway analysis by mRNA-sequencing. Drug efficacy studies were run with representative xenografts using the approved drug cetuximab as well as the new PI3K inhibitor PX-866. Tumors maintained their original morphology, genetic profiles and drug susceptibilities through serial passaging. The genetic makeup of these tumors was consistent with known frequencies of TP53, PI3KCA, NOTCH1 and NOTCH2 mutations. Because the EGFR inhibitor cetuximab is a standard HNSCC therapy, we tested its efficacy and observed a wide spectrum of efficacy. Cetuximab-resistant strains had higher PI3K/Akt pathway gene expression and protein activation than cetuximab-sensitive strains. The PI3K inhibitor PX-866 had anti-tumor efficacy in HNSCC models with PIK3CA alterations. Finally, PI3K inhibition was effective in two cases with NOTCH1 inactivating mutations. In summary, we have developed an HNSCC model covering its clinical spectrum whose major genetic alterations and susceptibility to anticancer agents represent contemporary HNSCC. This model enables to prospectively test therapeutic-oriented hypotheses leading to personalized medicine.

Background We have an incomplete understanding of the differences between cancer stem cells (CSCs) in human papillomavirus-positive (HPV-positive) and -negative (HPV-negative) head and neck squamous cell cancer (HNSCC). The PI3K pathway has the most frequent activating genetic events in HNSCC (especially HPV-positive driven), but the differential signaling between CSCs and non-CSCs is also unknown. Methods We addressed these unresolved questions using CSCs identified from 10 HNSCC patient-derived xenografts (PDXs). Sored populations were serially passaged in nude mice to evaluate tumorigenicity and tumor recapitulation. The transcription profile of HNSCC CSCs was characterized by mRNA sequencing, and the susceptibility of CSCs to therapy was investigated using an in vivo model. SOX2 transcriptional activity was used to follow the asymmetric division of PDX-derived CSCs. All statistical tests were two-sided. Results CSCs were enriched by high aldehyde dehydrogenase (ALDH) activity and CD44 expression and were similar between HPV-positive and HPV-negative cases (percent tumor formation injecting ≤ 1x10(3) cells: ALDH(+)CD44(high) = 65.8%, ALDH(-)CD44(high) = 33.1%, ALDH(+)CD44(high) = 20.0%; and injecting 1x10(5) cells: ALDH(-)CD44(low) = 4.4%). CSCs were resistant to conventional therapy and had PI3K/mTOR pathway overexpression (GSEA pathway enrichment, P < .001), and PI3K inhibition in vivo decreased their tumorigenicity (40.0%-100.0% across cases). PI3K/mTOR directly regulated SOX2 protein levels, and SOX2 in turn activated ALDH1A1 (P < .001 013C and 067C) expression and ALDH activity (ALDH(+) [%] empty-control vs SOX2, 0.4% ± 0.4% vs 14.5% ± 9.8%, P = .03 for 013C and 1.7% ± 1.3% vs 3.6% ± 3.4%, P = .04 for 067C) in 013C and 067 cells. SOX2 enhanced sphere and tumor growth (spheres/well, 013C P < .001 and 067C P = .04) and therapy resistance. SOX2 expression prompted mesenchymal-to-epithelial transition (MET) by inducing CDH1 (013C P = .002, 067C P = .01), followed by asymmetric division and proliferation, which contributed to tumor formation. Conclusions The molecular link between PI3K activation and CSC properties found in this study provides insights into therapeutic strategies for HNSCC. Constitutive expression of SOX2 in HNSCC cells generates a CSC-like population that enables CSC studies.

Mannose-binding lectin-associated serine proteases-1/3 (MASP-1/3) are essential in activating the alternative pathway (AP) of complement through cleaving pro-factor D (pro-Df) into mature Df. MASP are believed to require binding to mannose binding lectins (MBL) or ficolins (FCN) to carry out their biological activities. Murine sera have been reported to contain MBL-A, MBL-C, and FCN-A, but not FCN-B that exists endogenously in monocytes and is thought not to bind MASP-1. We examined some possible mechanisms whereby MASP-1/3 might activate the AP. Collagen antibody-induced arthritis, a murine model of inflammatory arthritis dependent on the AP, was unchanged in mice lacking MBL-A, MBL-C, and FCN-A (MBL(-/-)/FCN A(-/-) mice) in comparison to wild-type mice. The in vitro induction of the AP by adherent mAb to collagen II was intact using sera from MBL(-/-)/FCN A(-/-) mice. Furthermore, sera from MBL(-/-)/FCN A(-/-) mice lacked pro-Df and possessed only mature Df. Gel filtration of sera from MBL(-/-)/FCN A(-/-) mice showed the presence of MASP-1 protein in fractions containing proteins smaller than the migration of MBL-A and MBL-C in sera from C4(-/-) mice, suggesting possible binding of MASP-1 to an unknown protein. Lastly, we show that FCN-B was present in the sera of MBL(-/-)/FCN A(-/-) mice and that it was bound to MASP-1. We conclude that MASP-1 does not require binding to MBL-A, MBL-C, or FCN-A to activate the AP. MASP-1 may cleave pro-Df into mature Df through binding to FCN-B or to an unknown protein, or may function as an unbound soluble protein.

Gene-targeted mice deficient in the complement mannose-binding lectin-associated serine protease-1 and -3 (MASP1/3−/−) express only the zymogen of factor D (pro-factor D [pro-Df]), a necessary component of the alternative pathway (AP). We used the murine collagen Ab-induced arthritis (CAIA) model, in which the AP is unique among complement pathways in being both necessary and sufficient for disease induction, to determine whether MASP-1/3 are required in vivo for the development of tissue injury. Disease activity scores, complement C3 tissue deposition in the joint, and histopathologic injury scores were markedly decreased in MASP1/3−/− as compared with wild-type (WT) mice. MASP-1 protein was immunochemically localized to synovial cells of knees of WT mice with arthritis. Pro-Df was present in both synovial cells and chondrocytes of knees of WT and MASP1/3−/− mice without arthritis, with increased amounts present in synovial cells of WT mice with CAIA. No conversion of pro-Df to mature Df was detectable in the serum of MASP1/3−/− mice during the evolution of CAIA. C3 activation and deposition as well as C5a generation induced in vitro by adherent anti-type II collagen mAbs were absent using sera from MASP1/3−/− mice under conditions in which only the AP was active. The addition of human Df fully reconstituted in vitro C3 activation and C5a generation using sera from MASP1/3−/− mice. Our studies demonstrate for the first time, to our knowledge, the absolute requirement for the activity of MASP-1 protein in autoimmune-associated inflammatory tissue injury in vivo through activation of the AP of complement by cleavage of pro-Df to mature Df.

Summary Background This phase 1, dose-finding study determined the safety, maximum tolerated dose (MTD)/recommended phase 2 dose (RP2D), antitumor activity, and molecular correlates of IPI-926, a Hedgehog pathway (HhP) inhibitor, combined with cetuximab in patients with relapsed/metastatic squamous cell carcinoma of the head and neck. Patients and methods Cetuximab was given with a 400 mg/m 2 loading dose followed by 250 mg/m 2 weekly. IPI-926 was given daily starting two weeks after cetuximab initiation. A “3 + 3” study design was used. Prior therapy with cetuximab was allowed. Tumor biopsies occurred prior to cetuximab initiation, prior to IPI-926 initiation, and after treatment with both drugs. Results Nine patients were enrolled. The RP2D was 160 mg, the same as the single-agent IPI-926 MTD. Among 9 treated, 8 evaluable patients, the best responses were 1 partial response (12.5%), 4 stable disease (50%), and 3 disease progressions (37.5%). The median progression free survival was 77 days (95% confidence interval 39–156). Decreases in tumor size were seen in both cetuximab-naive patients (one HPV-positive, one HPV-negative). The most frequent treatment-emergent adverse events were fatigue, muscle cramps, and rash. No DLTs were observed. Tumor shrinkage and progression free survival were associated with intra-tumoral ErbB and HhP gene expression down-regulation during therapy, supporting the preclinical hypothesis. Conclusion Treatment with IPI-926 and cetuximab yielded expected toxicities with signs of anti-tumor activity. Serial tumor biopsies were feasible and revealed proof-of-concept biomarkers.

Local control and overall survival in patients with advanced head and neck squamous cell cancer (HNSCC) remains dismal. Signaling through the Hedgehog (Hh) pathway is associated with epithelial-to-mesenchymal transition, and activation of the Hh effector transcription factor Gli1 is a poor prognostic factor in this disease setting. Here, we report that increased GLI1 expression in the leading edge of HNSCC tumors is further increased by irradiation, where it contributes to therapeutic inhibition. Hh pathway blockade with cyclopamine suppressed GLI1 activation and enhanced tumor sensitivity to radiotherapy. Furthermore, radiotherapy-induced GLI1 expression was mediated in part by the mTOR/S6K1 pathway. Stroma exposed to radiotherapy promoted rapid tumor repopulation, and this effect was suppressed by Hh inhibition. Our results demonstrate that Gli1 that is upregulated at the tumor–stroma intersection in HNSCC is elevated by radiotherapy, where it contributes to stromal-mediated resistance, and that Hh inhibitors offer a rational strategy to reverse this process to sensitize HNSCC to radiotherapy. Cancer Res; 74(23); 7024–36. ©2014 AACR.

The dual pathway inhibitor rigosertib inhibits phosphoinositide 3-kinase (PI3K) pathway activation as well as polo-like kinase 1 (PLK1) activity across a broad spectrum of cancer cell lines. The importance of PIK3CA alterations in squamous cell carcinoma of the head and neck (HNSCC) has raised interest in exploring agents targeting PI3K, the product of PIK3CA. The genetic and molecular basis of rigosertib treatment response was investigated in a panel of 16 HNSCC cell lines, and direct patient tumor xenografts from eight patients with HNSCC [four HPV-serotype16 (HPV16)–positive]. HNSCC cell lines and xenografts were characterized by pathway enrichment gene expression analysis, exon sequencing, gene copy number, Western blotting, and immunohistochemistry (IHC). Rigosertib had potent antiproliferative effects on 11 of 16 HPV− HNSCC cell lines. Treatment sensitivity was confirmed in two cell lines using an orthotopic in vivo xenograft model. Growth reduction after rigosertib treatment was observed in three of eight HNSCC direct patient tumor lines. The responsive tumor lines carried a combination of a PI3KCA-activating event (amplification or mutation) and a p53-inactivating event (either HPV16- or mutation-mediated TP53 inactivation). In this study, we evaluated the in vitro and in vivo efficacy of rigosertib in both HPV+ and HPV− HNSCCs, focusing on inhibition of the PI3K pathway. Although consistent inhibition of the PI3K pathway was not evident in HNSCC, we identified a combination of PI3K/TP53 events necessary, but not sufficient, for rigosertib sensitivity. Mol Cancer Ther; 12(10); 1994–2005. ©2013 AACR.

The complement cascade is an important part of the innate immune system, but pathological activation of this system causes tissue injury in several autoimmune and inflammatory diseases, including immune complex glomerulonephritis. We examined whether mice with targeted deletion of the gene for factor B ( fB−/− mice) and selective deficiency in the alternative pathway of complement are protected from injury in the nephrotoxic serum (NTS) nephritis model of antibody-mediated glomerulonephritis. When the acute affects of the anti-glomerular basement membrane antibody were assessed, fB−/− mice developed a degree of injury similar to wild-type controls. If the mice were presensitized with sheep IgG or if the mice were followed for 5 mo postinjection, however, the fB−/− mice developed milder injury than wild-type mice. The immune response of fB−/− mice exposed to sheep IgG was similar to that of wild-type mice, but the fB−/− mice had less glomerular C3 deposition and lower levels of albuminuria. These results demonstrate that fB−/− mice are not significantly protected from acute heterologous injury in NTS nephritis but are protected from autologous injury in response to a planted glomerular antigen. Thus, although the glomerulus is resistant to antibody-initiated, alternative pathway-mediated injury, inhibition of this complement pathway may be beneficial in chronic immune complex-mediated diseases.

The complement system, especially the alternative pathway, plays essential roles in the induction of injury in collagen Ab-induced arthritis (CAIA) in mice. The goal of the current study was to directly compare the roles of receptors for C3a and C5a, as well as the membrane attack complex, as effector mechanisms in the pathogenesis of CAIA. Clinical disease activity in C3aR−/−, C5aR−/−, and C6-deficient (C6-def) mice was decreased by 52, 94, and 65%, respectively, as compared with wild-type mice. Decreases in histopathologic injury as well as in IgG and C3 deposition paralleled the clinical disease activity. A decrease in the percentage of synovial neutrophils was observed in C3aR−/−, C5aR−/−, and C6-def mice, and a decrease in macrophages was observed in C3aR−/− and C5aR−/−, but not in C6-def, mice. Synovial mRNA obtained by laser capture microdissection exhibited a decrease in TNF-α in C5aR−/− mice and in IL-1β in both C5aR−/− and C6-def mice, whereas C3aR−/− mice demonstrated no change in either cytokine. Our findings show that absent C3aR-, C5aR-, or membrane attack complex-initiated effector mechanisms each decrease susceptibility to CAIA, with clinical effects most pronounced in C5aR-deficient mice. Although the absence of C3aR, C5aR, or C6 led to differential deficiencies in effector mechanisms, decreased proximal joint IgG and C3 deposition was common to all three genotypes in comparison with wild-type mice. These data suggest the existence of positive-feedback amplification pathways downstream of all three effectors that promote additional IgG deposition and C3 activation in the joint.

Rheumatoid arthritis is associated with the development of autoantibodies to citrullinated self-proteins. Citrullinated synovial proteins, which are generated via the actions of the protein arginine deiminases (PADs), are known to develop in the murine collagen-induced arthritis (CIA) model of inflammatory arthritis. Given these findings, we evaluated whether N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-l-ornithine amide (Cl-amidine), a recently described pan-PAD inhibitor, could affect the development of arthritis and autoimmunity by treating mice in the CIA model with Cl-amidine on days 0–35. Cl-amidine treatment reduced total synovial and serum citrullination, decreased clinical disease activity by ∼50%, and significantly decreased IgG2a anti-mouse type II collagen Abs. Additionally, histopathology scores and total complement C3 deposition were significantly lower in Cl-amidine–treated mice compared with vehicle controls. Synovial microarray analyses demonstrated decreased IgG reactivity to several native and citrullinated epitopes compared with vehicle controls. Cl-amidine treatment had no ameliorative effect on collagen Ab-induced arthritis, suggesting its primary protective mechanism was not mediated through effector pathways. Reduced levels of citrullinated synovial proteins observed in mice treated with Cl-amidine are consistent with the notion that Cl-amidine derives its efficacy from its ability to inhibit the deiminating activity of PADs. In total, these results suggested that PADs are necessary participants in the autoimmune and subsequent inflammatory processes in CIA. Cl-amidine may represent a novel class of disease-modifying agents that modulate aberrant citrullination, and perhaps other immune processes, necessary for the development of inflammatory arthritis.

Objective The serum of most neuromyelitis optica (NMO) patients contains autoantibodies (NMO-IgGs) directed against the aquaporin-4 (AQP4) water channel located on astrocyte foot processes in the perivessel and subpial areas of the brain. Our objectives were to determine the source of central nervous system (CNS) NMO-IgGs and their role in disease pathogenesis. Methods Fluorescence-activated cell sorting and single-cell reverse transcriptase polymerase chain reaction were used to identify overrepresented plasma cell immunoglobulin (Ig) sequences in the cerebrospinal fluid (CSF) of an NMO patient after a first clinical attack. Monoclonal recombinant antibodies (rAbs) were generated from the paired heavy and light chain sequences and tested for target specificity and Fc effector function. The effect of CSF rAbs on CNS immunopathology was investigated by delivering single rAbs to rats with experimental autoimmune encephalomyelitis (EAE). Results Repertoire analysis revealed a dynamic, clonally expanded plasma cell population with features of an antigen-targeted response. Using multiple independent assays, 6 of 11 rAbs generated from CSF plasma cell clones specifically bound to AQP4. AQP4-specific rAbs recognized conformational epitopes and mediated both AQP4-directed antibody-dependent cellular cytotoxicity and complement-mediated lysis. When administered to rats with EAE, an AQP4-specific NMO CSF rAb induced NMO immunopathology: perivascular astrocyte depletion, myelinolysis, and complement and Ig deposition. Interpretation Molecular characterization of the CSF plasma cell repertoire in an early NMO patient demonstrates that AQP4-specfic Ig is synthesized intrathecally at disease onset and directly contributes to CNS pathology. AQP4 is now the first confirmed antigenic target in human demyelinating disease. Ann Neurol 2009;66:617–629

The alternative pathway (AP) of complement is required for the induction of collagen Ab-induced arthritis (CAIA) in mice. The objective of this study was to examine the effect of a recombinant AP inhibitor containing complement receptor 2 and factor H (CR2-fH) on CAIA in mice. CR2 binds to tissue-fixed activation fragments of C3, and the linked fH is a potent local inhibitor of the AP. CAIA was induced in C57BL/6 mice by i.p. injections of 4 mAb to type II collagen (CII) on day 0 and LPS on day 3. PBS or CR2-fH (250 or 500 μg) were injected i.p. 15 min after the mAb to CII on day 0 and 15 min after LPS on day 3; the mice were sacrificed on day 10. The disease activity score (DAS) was decreased significantly (p < 0.001) in both groups receiving CR2-fH compared with the PBS. Histology scores for inflammation, pannus, bone damage, and cartilage damage decreased in parallel with the DAS. C3 deposition in the synovium and cartilage was significantly reduced (p < 0.0001) in the mice treated with CR2-fH. In vitro studies with immune complexes containing type II collagen and mAb to CII showed that CR2-fH specifically inhibited the AP with minimal effect on the classical pathway (CP) and no effect on the lectin pathway (LP). The relative potency of CR2-fH in vitro was superior to mAbs to factor B and C5. Thus, CR2-fH specifically targets and inhibits the AP of complement in vitro and is effective in CAIA in vivo.