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Supplementary Figure 1 from KIT Pathway Alterations in Mucosal Melanomas of the Vulva and Other Sites

Purpose: A significant proportion of mucosal melanomas contain alterations in KIT. The aim of this study was to characterize the pattern of KIT, NRAS, and BRAF mutations in mucosal melanomas at specific sites and to assess activation of the KIT downstream RAF/MEK/extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/AKT pathways in mucosal melanoma specimens.Experimental Design: Seventy-one primary mucosal melanomas from various sites were studied. Mutation analysis was done by DNA sequencing. Expression of KIT, phosphorylated (p)-ERK, and p-AKT was evaluated by immunohistochemistry.Results: KIT mutations were detected in 35% (8 of 23) of vulvar, 9% (2 of 22) of anorectal, 7% (1 of 14) of nasal cavity, and 20% (1 of 5) of penile melanomas. No KIT mutations were found in 7 vaginal melanomas. The difference in KIT mutation frequency between vulvar and nonvulvar cases was statistically significant (P = 0.014). The overall frequencies of NRAS and BRAF mutations were 10% and 6%, respectively. Notably, vaginal melanomas showed a NRAS mutation rate of 43%. KIT gene amplification (≥4 copies), as assessed by quantitative real-time PCR, was observed in 19% of cases. KIT expression was associated with KIT mutation status (P < 0.001) and was more common in vulvar than nonvulvar tumors (P = 0.016). Expression of p-ERK and p-AKT was observed in 42% and 59% of tumors, respectively, and occurred irrespective of KIT/NRAS/BRAF mutation status. NRAS mutation was associated with worse overall survival in univariate analysis.Conclusions: Results show that KIT mutations are more common in vulvar melanomas than other types of mucosal melanomas and that both the RAF/MEK/ERK and PI3K/AKT pathways are activated in mucosal melanoma specimens. Clin Cancer Res; 17(12); 3933–42. ©2011 AACR.

Background: Resistance to endocrine therapy is a clinical problem and is not explained by lack or loss of the oestrogen receptor (ER). Up-regulation of several receptor tyrosine kinases have been correlated to diminished effect of endocrine treatment. More contradictory are the role of downstream mitogen activated protein kinases (MAPK's). Patients and Methods: We performed a tissue micro array (TMA's) on patients that originate from an earlier presented population of 404 patients with a steroid receptor positive BC subjected to tamoxifen as only adjuvant systemic treatment. Material from 120 patients was available for the TMA. JNK, p38 and ERK was determined by use of immunohistochemistry and scored 0, +1, +2, +3. Patients were grouped into two categories for statistical analyses. Results: Absence or low JNK was statistically significantly correlated with HER2+ (p=0.018), a Luminal B like subtype (ER+, high proliferation and/or HER2+) (p=0.007), shorter relapse-free (p= 0.0494) and breast cancer corrected survival (p=0.0458). High ERK was more frequent among node-negative patients (p=0.00469) and more frequent in relapse-free patients; RFS (p= 0.225); BCCS (p= 0.0705). High expression of p38 was associated with p38 previously determined by ELISA (p=0.085) and HER2+ (p=0.094), however not reaching statistical significance. High p38 was related to increased relapses within the first years but the difference vanished with longer follow-up time; RFS (p=0.486) and BCCS (p= 0.589). JNK remained as an independent marker of RFS in a Cox proportional multivariate analysis (HR=2.19; 95%CI=1.14−11.41; p=0.029), together with nodal status (HR=3.6; 95%CI=1.44−8.81; p=0.006) and histological grade (HR=3.1; 95%CI=1.29−7.56; p=0.011). Discussion: Our data on ERK and p38 is concordant with results from previous published studies. Two studies have shown ERK expression, determined by IHC to be correlated with “good prognosis features” as smaller tumour size, absence of nodal metastasis, lower proliferation and a favourable outcome after adjuvant tamoxifen (Svensson et. al. 2005, Bergqvist et. al. 2006). Higher p38 determined by use of an ELISA has been suggested as a marker of early relapses i.e. intrinsic resistance, although methodological problems must be taken into account (Linderholm et. al. 2011). By use of IHC we could confirm that patients with higher expression of p38 actually had significantly more relapses within early follow-up. JNK was the MAPK with the most pronounced effect on survival. Absent or low expression was significantly correlated with an impaired survival in both uni- and multivariate analyses, a finding to our very best knowledge not described before. Low JNK was actually found more frequently in patients with a HER2 positive or Luminal B like BC (ER+, high proliferation rate or HER2+). Conclusions: Our results suggest that MAPK's can be evaluated by IHC on TMA's. Absence/low JNK was significantly correlated with an impaired survival and a Luminal B like subtype. Confirming studies in large preferable randomised patient populations are warranted. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-19.

For clinically relevant studies on melanoma progression and invasiveness, in vivo experimental systems with a human cellular microenvironment would be advantageous. We have compared tumor formation from a human cutaneous malignant melanoma cell line (BL), after injection as conventional xenografts in the mouse, or when injected into a predominantly species-specific environment of human embryonic stem cell–derived teratoma induced in the mouse (the hEST model). The resulting melanoma histology was generally analogous, both systems showing delimited densely packed areas with pleomorphic cells of malignant appearance. A specificity of the integration process into the human embryonic teratoma tissues was indicated by the melanoma exclusively being found in areas compatible with condensed mesenchyme, similar to neural crest development. Here, also enhanced neovascularization was seen within the human mesenchymal tissues facing the BL melanoma growth. Furthermore, in the hEST model an additional melanoma cell phenotype occurred, located at the border of, or infiltrating into, the surrounding human loose mesenchymal fibrous stroma. This BL population had a desmoplastic spindle-like appearance, with markers indicative of dedifferentiation and migration. The appearance of this apparently more aggressive phenotype, as well as the induction of human angiogenesis, shows specific interactions with the human embryonic microenvironment in the hEST model. In conclusion, these data provide exciting options for using the hEST model in molecular in vivo studies on differentiation, invasiveness, and malignancy of human melanoma, while analyzing species-specific reactions in vivo. [Cancer Res 2009;69(9):3746–54]

Acute myeloid leukemia (AML) is a hematological malignancy characterized by a clonal proliferation of myeloid precursor cells. Acquired genetic aberrations have been linked to leukemogenic events such as blocked differentiation and deregulated proliferation. Conventional chemotherapeutic regimens used for AML typically involve anthracyclines and cytarabine, often resulting in complete remission (CR), although relapsing disease with a resistant phenotype is still common. Thus, novel therapeutic strategies are clearly needed. We have shown that the CD33-targeted drug gemtuzumab ozogamicin (GO) can induce significant DNA double strand break (dsbs) formation and activate pro-apoptotic signaling via Bak, Bax, and Caspase-3 in AML. The interest in using “old” FDA-approved drugs for new indications is increasing. We have previously reported that phenothiazines, compounds in clinical use for psychiatric disorders, have the capacity to increase chemotherapy-induced cell death by increasing DNA damage signaling and block DNA repair in solid tumor cells. Here we evaluated the effect of phenothiazines as mono therapy, and in combination with different classes of chemotherapeutics, on AML cells representing different leukemic subtypes. A panel of structurally different phenothiazines (trifluoperazine, thiothixene, chlorpromazine, triflupromazine, fluphenazine, cis-flupenthixol, prochlorperazine, and hydroxyzine) was evaluated for induction of in vitro cytotoxicity in the AML cell lines HL60, Kasumi-1, KG1a, and NB4, representing different AML subtypes. When used as mono therapy trifluoperazine (TFP) was the most efficient cell death inducing phenothiazine. Next the effect of phenothiazines on chemotherapy-induced responses was examined. A sub toxic dose of TFP, causing a 10% reduction in cell viability, was used in combination with the chemotherapeutics daunorubicin or GO. Pre-incubating AML cells for 1h with TFP increased GO-induced cytoxicity by up to 45%. Interestingly, a greater sensitization of chemotherapy-induced cell kill was observed in GO-resistant Kasumi-1 cells than in responsive HL-60 cells. In line with previous results in solid tumor malignancies we found that TFP increased chemotherapy-induced DNA damage signaling, Bak/Bax and caspase-3 activation also in the AML cells examined, suggesting increased DNA damage signaling to be operative. In conclusion, we show that AML cells are differentially sensitive to a panel of phenothiazines when used in mono therapy and can potentiate different classes of chemotherapeutics by increasing DNA damage response signaling. Thus, this may be a promising treatment option for AML which needs to be further explored by analyzing AML patient derived cells, studies which are ongoing within our group. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B19. Citation Format: Petra Haag, Katarzyna Zielinska-Chomej, Therese Juntti, Lena Kanter, Rolf Lewensohn, Leif Stenke, Kristina Viktorsson. Phenothiazines induce cytotoxicity and enhance chemotherapy-induced cell death signaling in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B19.

Acute myeloid leukemia (AML) is characterized by uncontrolled expansion of immature leukocytes. It is the predominant leukemia among adults and is generally treated with high dose chemotherapy. This normally results in an initial normalization of the blood- and bone marrow-cell composition (complete remission; CR). Yet, most patients relapse within 1–2 years. Conventional chemotherapy is also associated with severe and at times lethal side effects. Thus, it would be valuable to find both biomarkers capable of predicting treatment response and novel treatment strategies. Here we address both these issues. Identification of biomarkers to predict CR was analyzed by gene expression analysis of AML patient cells. To further understand the potential of gemtuzumab ozogamicin (GO) as targeted agent for AML, profiling of apoptotic signaling in AML patient derived cells and cell lines was performed. Cellular signaling was compared to the conventional drug Daunorubicin. For the biomarker study, gene expression profiling (Affymetrix U133A) was made on leukemic blast cells isolated from AML patients (n=42) with long (> 6 months).or short (< 6 months) CR time. Candidate genes were validated using real time quantitative PCR in patient cohorts and individual samples. Ingenuity pathway analysis was applied to sort out critical signaling components involved. The molecular studies of GO response were focused on caspase-2. The AML cell line HL60 was treated in vitro with GO or Daunorubicin with or without caspase-2 inhibitor z-VDVAD. Patient derived blasts were treated in vitro with GO or were untreated. Caspase-2 expression and processing was analyzed using western blotting. Caspase-3, Bak/Bax-activation assays and cell cycle profiling was analyzed using FACS. We found major discrepancies in gene expression when comparing AML patients with long or short CR time. RUNX1T1 is frequently translocated with RUNX1 to produce the characteristic fusion gene (t8;21) in AML and is blocking hematopoietic differentiation. Here we found the transcription factor RUNX1T1 (ETO) to be highly over expressed in patients with short CR duration. Examples of other up-regulated genes in patients with short CR were TKTL1, NUDT4 and CHD3. A number of genes that were lower expressed in patients with short CR were ANXA1, FLRT3 and TLR8. We have molecularly dissected the role of caspase-2 in GO-induced apoptotic signaling and compared its mechanisms of action to Daunorubicin. We found processing of caspase-2 in both GO- and Daunorubicin-induced apoptotic signaling in AML cells and after GO-treatment in patient derived leukemic blasts. A critical role of caspase-2 in GO-induced apoptosis was found as the caspase-2 inhibitor z-VDVAD-fmk reduced both caspase-3 activation and apoptotic morphology with about 50% yet having no effect on GO-induced Bak and Bax activation. These results suggest that caspase-2 is located upstream of caspase-3, in this signaling cascade. In order to further understand the clinical relevance of our findings, we are currently analyzing expression levels of caspase-2 and caspase-3 in patient cells. In conclusion, our gene expression profiling suggest RUNX1T1 to be a marker of CR duration in AML and our molecular profiling suggest caspase-2 to have a critical role in CT response of AML. Thus both these analyzes highlight possible CT resistance mechanisms in AML which warrants further studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A24.

We are investigating signaling molecules involved in apoptotic cell death in acute myeloid leukemia (AML) cells after treatment with the targeted drug gemtuzumab ozogamicin (GO) and the conventional drug daunorubicin, with focus on caspase-2. New effective therapeutic drugs are highly desirable in the treatment of AML. Although a majority of treated patients initially respond to conventional treatment, most patients will relapse within 1-2 years. Also, the treatment is associated with severe off-target effects. GO is a novel therapeutic approach, consisting of a monoclonal CD33 antibody coupled to calicheamicin, which cause DNA double strand breaks (DSB). We demonstrated that activation of Bak and Bax is critical for GO- and daunorubicin- induced apoptotic signaling in AML cells. The activation was followed by mitochondrial depolarization, caspase-3 activation and nuclear fragmentation. We continued to examine potential proapoptotic signaling events upstream of mitochondria in HL60 AML cells in vitro. We identified caspase-2 and the pro-apoptotic BH3-only protein Bid as two candidates. By analyzing caspase-2 by western blotting after GO treatment we found that GO caused cleavage of caspase-2 after 24h, which was prior to mitochondria-mediated signaling. Also, full-length Bid was cleaved to its truncated, active form tBid. The importance of caspase-2 activation in the signaling cascade after GO treatment was demonstrated using z-VDVAD-fmk, a caspase-2 inhibitor. Preincubation with this inhibitor reduced caspase-3 activation with around 50%, suggesting that caspase-2 is an important molecule, located upstream of caspase-3, in the signaling between DNA double stranded breaks and apoptosis signaling in AML cells. In conclusion, we demonstrate that GO and daunorubicin, at clinically applicable concentrations, induce pro-apoptotic signaling which involve activation of caspase-2, and activation of the BH3-only protein Bid. Blocking caspase-2 activity reduced caspase-3 activation with 50%. This suggests multiple signaling pathways capable of activating caspase-3. The caspase-2 pathway appears central for downstream caspase-3 activity, after GO treatment in AML. Our findings may highlight possible resistance mechanisms in AML, which might have profound therapeutic implications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4090. doi:10.1158/1538-7445.AM2011-4090

The aim of this study was to identify essential signaling molecules involved in apoptotic cell death in acute myeloid leukemia (AML) cells after treatment with the targeted drug gemtuzumab ozogamicin (GO). New effective therapeutic drugs are highly desirable in the treatment of AML. Although a majority of treated patients initially respond to conventional treatment, most patients will relapse within 1–2 years. Also, the treatment is associated with severe off-target effects. GO is a novel therapeutic approach, consisting of a monoclonal CD33 antibody coupled to a DNA damaging toxin, calicheamicin. GO enters the cell after binding to the CD33 antigen, preferentially expressed on myeloid cells, and cause DNA double strand breaks (DSB). We have previously demonstrated that activation of Bak and Bax is critical for GO-induced apoptotic signaling, followed by downstream mitochondrial depolarization and caspase-3 activation. As a continuation, we have now examined potential proapoptotic signaling events induced by GO upstream of mitochondria in HL60 AML cells in vitro. We identified caspase-2 and the pro-apoptotic BH3-only protein Bid as two candidates. By analyzing caspase-2 by western blotting after GO treatment we found that GO caused cleavage of caspase-2 after 24h, which was prior to mitochondria-mediated signaling. At the same time, full-length Bid was cleaved to its truncated, active form tBid. The importance of caspase-2 activation in the signaling cascade after GO treatment was demonstrated using z-VDVAD-fmk, a caspase 2 inhibitor. Preincubation with this inhibitor reduced caspase-3 activation with around 50%, suggesting that caspase-2 is an important molecule, located upstream of caspase-3, in the signaling between DNA double stranded breaks and apoptosis signaling in AML cells. In conclusion, we demonstrate that GO, at clinically applicable concentrations (100–1000ng/ml), induce pro-apoptotic signaling which involve activation of caspase-2, and cleavage of the BH3-only protein Bid to the truncated, active protein tBid. Blocking caspase-2 activity reduced caspase-3 activation to only half. This suggests multiple signaling pathways capable of activating caspase-3. The caspase-2 pathway appears central for downstream caspase-3 activity, after GO treatment in AML. Our findings may highlight possible resistance mechanisms in AML, which might have profound therapeutic implications. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B25.