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Reversible inhibition of lysine specific demethylase 1 is a novel anti-tumor strategy for poorly differentiated endometrial carcinoma.

Theisen ER, Gajiwala S, Bearss J, Sorna V, Sharma S, Janat-Amsbury M - BMC Cancer (2014)

Bottom Line: These effects were largely dose-dependent.Orthotopic implantation of KLE cells resulted in slow-growing and diffuse tumors throughout the abdomen.Treatment with HCI2509 resulted 5/9 tumor regressions such that treatment and regressions were significantly associated (p=0.034).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, University of Utah, Salt Lake City, UT, USA. margit.janat-amsbury@hsc.utah.edu.

ABSTRACT

Background: Endometrial cancer is the most common gynecologic malignancy. Type II endometrial carcinoma is often poorly differentiated and patients diagnosed with Type II disease (~11%) are disproportionately represented in annual endometrial cancer deaths (48%). Recent genomic studies highlight mutations in chromatin regulators as drivers in Type II endometrial carcinoma tumorigenesis, suggesting the use of epigenetic targeted therapies could provide clinical benefit to these patients. We investigated the anti-tumor efficacy of the LSD1 inhibitor HCI2509 in two poorly differentiated Type II endometrial cancer cell lines AN3CA and KLE.

Methods: The effects of HCI2509 on viability, proliferation, anchorage-independent growth, global histone methylation, LSD1 target gene induction, cell cycle, caspase activation and TUNEL were assayed. KLE cells were used in an orthotopic xenograft model to assess the anti-tumor activity of HCI2509.

Results: Both AN3CA and KLE cells were sensitive to HCI2509 treatment with IC50s near 500 nM for cell viability. Inhibition of LSD1 with HCI2509 caused decreased proliferation and anchorage independent growth in soft agar, elevated global histone methylation, and perturbed the cell cycle in both cell lines. These effects were largely dose-dependent. HCI2509 treatment also caused apoptotic cell death. Orthotopic implantation of KLE cells resulted in slow-growing and diffuse tumors throughout the abdomen. Tumor burden was distributed log-normally. Treatment with HCI2509 resulted 5/9 tumor regressions such that treatment and regressions were significantly associated (p=0.034).

Conclusions: Our findings demonstrate the anti-cancer properties of the LSD1 inhibitor HCI2509 on poorly differentiated endometrial carcinoma cell lines, AN3CA and KLE. HCI2509 showed single-agent efficacy in orthotopic xenograft studies. Continued studies are needed to preclinically validate LSD1 inhibition as a therapeutic strategy for endometrial carcinoma.

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HCI2509 treatment causes tumor regressionin vivo. (A) Average total body weight (g) of mice in both groups, vehicle and HCI2509 treatment, starting at implantation (day 0) through the course of the study. Data points shown represent the mean and standard deviation. (B) Quantified bioluminscence measurements of both the vehicle and HCI2509 treatment groups. Data is plotted as the geometric mean of total flux (photons/second). Daily treatment was initiated on day 7 (day 0 = implantation), such that day 14 represents the first day of imaging after the start of treatment. (C) Individual mouse images from study day 42 (day 35 of treatment). All images are on the same luminescence scale from 1.54 × 104 p/s to 8.66 × 106 p/s. Note: Animal #6 in the treatment group was sacrificed on study day 36, therefore the image is from study day 35 imaging.
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Fig5: HCI2509 treatment causes tumor regressionin vivo. (A) Average total body weight (g) of mice in both groups, vehicle and HCI2509 treatment, starting at implantation (day 0) through the course of the study. Data points shown represent the mean and standard deviation. (B) Quantified bioluminscence measurements of both the vehicle and HCI2509 treatment groups. Data is plotted as the geometric mean of total flux (photons/second). Daily treatment was initiated on day 7 (day 0 = implantation), such that day 14 represents the first day of imaging after the start of treatment. (C) Individual mouse images from study day 42 (day 35 of treatment). All images are on the same luminescence scale from 1.54 × 104 p/s to 8.66 × 106 p/s. Note: Animal #6 in the treatment group was sacrificed on study day 36, therefore the image is from study day 35 imaging.

Mentions: We further evaluated the efficacy of HCI2509 in an orthotopic xenograft model of endometrial carcinoma utilizing the KLE cell line stably transfected with luciferase to facilitate bioluminescence imaging. After implantation (day 0) and recovery, bioluminescence was measured weekly for the duration of the study (42 d). Total body weight was measured 3 times weekly, and weekly points were plotted (Figure 5A). At day 7, animals with detectable tumor were randomized into vehicle only and HCI2509 treatment groups (Additional file 4: Figure S5A). We observed the tumor luminescence values were better fit to a log-normal distribution than a normal distribution, which is common for various biological phenomena such as latency times for infections or survival times after a diagnosis of cancer (Additional file 4: Figure S5B) [44]. For this reason, the geometric mean of the tumor volumes for both conditions are plotted (Figure 5B). Values observed at day 7 were higher than those observed for the remainder of the study and therefore excluded from the graph. This initial burst of proliferation, and associated luminescence, followed by a drop off before later hitting exponential growth is commonly observed in xenograft studies. After 35 days of treatment (day 42 of the study) proliferating disease was observed in all of the vehicle treated animals, while 5 of the 9 drug treated animals showed no detectable luminescence (Figure 5C). Lack of luminescence is incorporated as the background reading of the instrument for each day of the experiment, as determined by an unimplanted, non-tumor bearing, healthy control. We used a Fisher’s exact test to evaluate the effect of treatment vs. vehicle on either tumor or regression and found HCI2509 significantly associated with tumor regression (p = 0.034). No difference in body weight was seen between the vehicle and treatment groups indicating tolerability of HCI2509. The luminescence readout for the untreated control group are plotted together with data from the vehicle and treatment groups in Additional file 4: Figure S5D as are the body weight measurements including the non-tumor bearing control. When considered with the in vitro data suggesting decreased proliferation, transformation and induced apoptosis in concert with increased global histone methylation and LSD1 engagement, these data support LSD1 inhibition with HCI2509 as a potential therapeutic strategy for Type II endometrial carcinoma.Figure 5


Reversible inhibition of lysine specific demethylase 1 is a novel anti-tumor strategy for poorly differentiated endometrial carcinoma.

Theisen ER, Gajiwala S, Bearss J, Sorna V, Sharma S, Janat-Amsbury M - BMC Cancer (2014)

HCI2509 treatment causes tumor regressionin vivo. (A) Average total body weight (g) of mice in both groups, vehicle and HCI2509 treatment, starting at implantation (day 0) through the course of the study. Data points shown represent the mean and standard deviation. (B) Quantified bioluminscence measurements of both the vehicle and HCI2509 treatment groups. Data is plotted as the geometric mean of total flux (photons/second). Daily treatment was initiated on day 7 (day 0 = implantation), such that day 14 represents the first day of imaging after the start of treatment. (C) Individual mouse images from study day 42 (day 35 of treatment). All images are on the same luminescence scale from 1.54 × 104 p/s to 8.66 × 106 p/s. Note: Animal #6 in the treatment group was sacrificed on study day 36, therefore the image is from study day 35 imaging.
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Fig5: HCI2509 treatment causes tumor regressionin vivo. (A) Average total body weight (g) of mice in both groups, vehicle and HCI2509 treatment, starting at implantation (day 0) through the course of the study. Data points shown represent the mean and standard deviation. (B) Quantified bioluminscence measurements of both the vehicle and HCI2509 treatment groups. Data is plotted as the geometric mean of total flux (photons/second). Daily treatment was initiated on day 7 (day 0 = implantation), such that day 14 represents the first day of imaging after the start of treatment. (C) Individual mouse images from study day 42 (day 35 of treatment). All images are on the same luminescence scale from 1.54 × 104 p/s to 8.66 × 106 p/s. Note: Animal #6 in the treatment group was sacrificed on study day 36, therefore the image is from study day 35 imaging.
Mentions: We further evaluated the efficacy of HCI2509 in an orthotopic xenograft model of endometrial carcinoma utilizing the KLE cell line stably transfected with luciferase to facilitate bioluminescence imaging. After implantation (day 0) and recovery, bioluminescence was measured weekly for the duration of the study (42 d). Total body weight was measured 3 times weekly, and weekly points were plotted (Figure 5A). At day 7, animals with detectable tumor were randomized into vehicle only and HCI2509 treatment groups (Additional file 4: Figure S5A). We observed the tumor luminescence values were better fit to a log-normal distribution than a normal distribution, which is common for various biological phenomena such as latency times for infections or survival times after a diagnosis of cancer (Additional file 4: Figure S5B) [44]. For this reason, the geometric mean of the tumor volumes for both conditions are plotted (Figure 5B). Values observed at day 7 were higher than those observed for the remainder of the study and therefore excluded from the graph. This initial burst of proliferation, and associated luminescence, followed by a drop off before later hitting exponential growth is commonly observed in xenograft studies. After 35 days of treatment (day 42 of the study) proliferating disease was observed in all of the vehicle treated animals, while 5 of the 9 drug treated animals showed no detectable luminescence (Figure 5C). Lack of luminescence is incorporated as the background reading of the instrument for each day of the experiment, as determined by an unimplanted, non-tumor bearing, healthy control. We used a Fisher’s exact test to evaluate the effect of treatment vs. vehicle on either tumor or regression and found HCI2509 significantly associated with tumor regression (p = 0.034). No difference in body weight was seen between the vehicle and treatment groups indicating tolerability of HCI2509. The luminescence readout for the untreated control group are plotted together with data from the vehicle and treatment groups in Additional file 4: Figure S5D as are the body weight measurements including the non-tumor bearing control. When considered with the in vitro data suggesting decreased proliferation, transformation and induced apoptosis in concert with increased global histone methylation and LSD1 engagement, these data support LSD1 inhibition with HCI2509 as a potential therapeutic strategy for Type II endometrial carcinoma.Figure 5

Bottom Line: These effects were largely dose-dependent.Orthotopic implantation of KLE cells resulted in slow-growing and diffuse tumors throughout the abdomen.Treatment with HCI2509 resulted 5/9 tumor regressions such that treatment and regressions were significantly associated (p=0.034).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, University of Utah, Salt Lake City, UT, USA. margit.janat-amsbury@hsc.utah.edu.

ABSTRACT

Background: Endometrial cancer is the most common gynecologic malignancy. Type II endometrial carcinoma is often poorly differentiated and patients diagnosed with Type II disease (~11%) are disproportionately represented in annual endometrial cancer deaths (48%). Recent genomic studies highlight mutations in chromatin regulators as drivers in Type II endometrial carcinoma tumorigenesis, suggesting the use of epigenetic targeted therapies could provide clinical benefit to these patients. We investigated the anti-tumor efficacy of the LSD1 inhibitor HCI2509 in two poorly differentiated Type II endometrial cancer cell lines AN3CA and KLE.

Methods: The effects of HCI2509 on viability, proliferation, anchorage-independent growth, global histone methylation, LSD1 target gene induction, cell cycle, caspase activation and TUNEL were assayed. KLE cells were used in an orthotopic xenograft model to assess the anti-tumor activity of HCI2509.

Results: Both AN3CA and KLE cells were sensitive to HCI2509 treatment with IC50s near 500 nM for cell viability. Inhibition of LSD1 with HCI2509 caused decreased proliferation and anchorage independent growth in soft agar, elevated global histone methylation, and perturbed the cell cycle in both cell lines. These effects were largely dose-dependent. HCI2509 treatment also caused apoptotic cell death. Orthotopic implantation of KLE cells resulted in slow-growing and diffuse tumors throughout the abdomen. Tumor burden was distributed log-normally. Treatment with HCI2509 resulted 5/9 tumor regressions such that treatment and regressions were significantly associated (p=0.034).

Conclusions: Our findings demonstrate the anti-cancer properties of the LSD1 inhibitor HCI2509 on poorly differentiated endometrial carcinoma cell lines, AN3CA and KLE. HCI2509 showed single-agent efficacy in orthotopic xenograft studies. Continued studies are needed to preclinically validate LSD1 inhibition as a therapeutic strategy for endometrial carcinoma.

Show MeSH
Related in: MedlinePlus