CRISPR CAS9 mediated BRCA1 knockout modulates …...infiltrating lymphocyte (TIL) population and the...
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CRISPR CAS9 mediated BRCA1 knockout modulates the tumor infiltrating lymphocytes landscape and cytokine profile in a murine breast cancer model Lashuk, Kanstantsin 1 ; Avrutskaya, Anya 2 ; Tschuch, Cordula 1 ; Jensen, Astrid 3 ; Heins, Mariette 4 ; Kelter, Gerhard 1 ; Peille, Anne-Lise 1 ; Robinson, Maycee 2 ; Durham, William 2 ; Maier, Armin 1 ; Zuurmond, Anne-Marie 3 ; Schüler, Julia 1 1 Charles River Research Services Germany GmbH Freiburg, Germany 2 Discovery Services Charles River Morrisville, NC 27560 3 Charles River, CR Leiden, The Netherlands 4 Charles River, CR Groningen, The Netherlands 2 RESULTS The creation of a murine breast cancer cell line bearing a homozygous frame shift mutation was successfully conducted. The comparison of the mutated vs the wt EMT6 cell line in vivo revealed significant differences in the tumor doubling time. The mutated cell line grew significantly slower (1.96 ± 0.38 vs 1.61 ± 0.37 d). The histological architecture was similar in both lines, depicting an undifferentiated carcinoma. The percentage of tumor infiltrating lymphocytes (TILs) was similar for CD45 (determined by FC and IHC). The subtyping of TILs revealed higher percentages for gMDSC and M2 macrophages in the EMT6 BRCA1 KO line. Nevertheless, those differences were not statistically significant. The cytokine profile of the two lines differed significantly with higher cytokine levels of 22/23 analytes in the non-modified cell line. G-CSF is the only determined cytokine expressing higher levels in the serum of EMT6 BRCA1 KO animals. Talazoparib was the most active compound in the EMT6 BRCA1 KO model, followed by Niraparib and Rucaparib. Olaparib was considered inactive. The EMT6 model was resistant against all tested PARPi. The EMT6 BRCA1 KO model turned out to be sensitive towards anti CTLA-4 treatment but showed mild tumor growth delay under anti PD-1 treatment in monotherapy. The EMT6 model was sensitive towards both checkpoint inhibitor treatments. Combination therapy was more effective in all tested settings. However, Rucaparib + anti CTLA-4 as well as Talazoparib + anti-PD-1 induced a significant prolongation of the life span of the treated animals. In the EMT6/BRCA1 KO the TIL population decreased under PARP inhibitor monotherapy (Talazoparib) but increased significantly under treatment with CP inhibitors anti CTLA-4 and anti PD-1 in monotherapy as well as in combination with Talazoparib (Kruskall-Wallis test, p< 0.005). In contrast, the TIL frequency in the EMT6 model was similar in all treatment arms. Differences in the TIL composition were mainly detected in the NK and gMDSC cell fraction which were upregulated under treatment including Talazoparib specifically in the EMT6/BRCA1 KO model. The secreted cytokine profile supported the TIL data by upregulation of multiple pro-inflammatory cytokines specifically in the EMT6 BRCA1 KO line. In line with the enhanced NK cell numbers under therapy was the fact that serum levels of RANTES and IL-2 were enhanced under treatment in EMT6/BRCA1 KO bearing animals. 1 INTRODUCTION Around 10% of breast cancer cases are attributed to genetic disorders like mutations in BRCA-1/2 genes. Targeted therapy of BRCA-deficient cancers has been achieved using poly(ADP-ribose) polymerase (PARP) inhibitors, which block BRCA-independent DNA repair. To study the effect of this common mutation on tumor biology in general and specifically the immunological landscape of the tumor microenvironment, we knocked out the BRCA1 gene in the murine EMT6 breast cancer cell line. Subsequently, we analyzed the tumor infiltrating lymphocyte (TIL) population and the cytokine profiles in serum of tumor bearing animals. 3 CONCLUSION Further mechanistic studies as well as comparative studies will elucidate the tumor biology behind these observations and might lead to beneficial combination strategies in patients suffering from triple negative and BRCA1-/- breast cancer Figure 1. Mode of action of PARP (A) and checkpoint (B) inhibitors and treatment regimen in combination (C) Fig 2. Creation of homozygous EMT6 BRCA1 KO line. Generation of a Brca1 knock-out in mouse breast cancer cell line EMT6 mice based on CRISPR technology using the HDR pathway SITC 2019 Annual Meeting, Nov 6 – 10 2019; National harbor , Maryland Figure 3. Basic tumor biology characteristics. The volume of individual tumors was determined twice weekly from the day of implantation on. TIL and cytokine analysis was performed at a tumor volume of 1500 mm³. A B A Mittica G. et al: Int J Mol Sci. 2016 Jul 20;17(7). pii: E1169. doi: 10.3390/ijms17071169. C Figure 5. Sensitivity towards PARP and checkpoint inhibitors in combination therapy. Tumor volume over time Figure 4. Sensitivity towards PARP and checkpoint inhibitors in monotherapy. monotherapy. The EMT6 model was sensitive towards both checkpoint inhibitor treatments. Combination therapy was more effective in all tested settings. However, Rucaparib + anti CTLA prolongation of the life span of the treated animals. volume over time Figure 6. Sensitivity towards PARP and checkpoint inhibitors in combination therapy. Overall survival Figure 7. Sensitivity towards PARP and checkpoint inhibitors in combination therapy in EMT6 BRCA1 KO (A) and EMT6 (B). TIL analysis B A Figure 8. Sensitivity towards PARP and checkpoint inhibitors in combination therapy in EMT6 BRCA1 KO (A) and EMT6 (B). Cytokine profile A B P524
Transcript of CRISPR CAS9 mediated BRCA1 knockout modulates …...infiltrating lymphocyte (TIL) population and the...
CRISPR CAS9 mediated BRCA1 knockout modulates the tumor infiltrating lymphocytes landscape and cytokine profile in a murine breast cancer model
Lashuk, Kanstantsin1; Avrutskaya, Anya2; Tschuch, Cordula1; Jensen, Astrid3; Heins, Mariette4 ; Kelter, Gerhard1; Peille, Anne-Lise1; Robinson, Maycee2; Durham, William2; Maier, Armin1; Zuurmond, Anne-Marie3; Schüler, Julia1
1 Charles River Research Services Germany GmbH Freiburg, Germany 2 Discovery Services Charles River Morrisville, NC 275603 Charles River, CR Leiden, The Netherlands4 Charles River, CR Groningen, The Netherlands
2 RESULTS The creation of a murine breast cancer cell line bearing a homozygous frame shift mutation was successfully conducted. The comparison of the mutated vs the wt EMT6 cell line in vivo revealed significant differences in the tumor doubling time. The mutated cell line grew significantly
slower (1.96 ± 0.38 vs 1.61 ± 0.37 d). The histological architecture was similar in both lines, depicting an undifferentiated carcinoma. The percentage of tumor infiltrating lymphocytes (TILs) was similar for CD45 (determined by FC and IHC). The subtyping of TILs revealed higher percentages for gMDSC and M2 macrophages in the EMT6 BRCA1 KO line. Nevertheless, those differences were not
statistically significant. The cytokine profile of the two lines differed significantly with higher cytokine levels of 22/23 analytes in the non-modified cell line. G-CSF is the only determined
cytokine expressing higher levels in the serum of EMT6 BRCA1 KO animals. Talazoparib was the most active compound in the EMT6 BRCA1 KO model, followed by Niraparib and Rucaparib. Olaparib was considered inactive. The EMT6 model was resistant against all tested PARPi. The EMT6 BRCA1 KO model turned out to be sensitive towards anti CTLA-4 treatment but showed mild tumor growth delay under anti PD-1 treatment in
monotherapy. The EMT6 model was sensitive towards both checkpoint inhibitor treatments. Combination therapy was more effective in all tested settings. However, Rucaparib + anti CTLA-4 as well as Talazoparib + anti-PD-1 induced a significant
prolongation of the life span of the treated animals. In the EMT6/BRCA1 KO the TIL population decreased under PARP inhibitor monotherapy (Talazoparib) but increased significantly under treatment with CP inhibitors
anti CTLA-4 and anti PD-1 in monotherapy as well as in combination with Talazoparib (Kruskall-Wallis test, p< 0.005). In contrast, the TIL frequency in the EMT6 model was similar in all treatment arms. Differences in the TIL composition were mainly detected in the NK and gMDSC cell fraction which were upregulated under treatment including Talazoparib specifically
in the EMT6/BRCA1 KO model. The secreted cytokine profile supported the TIL data by upregulation of multiple pro-inflammatory cytokines specifically in the EMT6 BRCA1 KO line. In line with the
enhanced NK cell numbers under therapy was the fact that serum levels of RANTES and IL-2 were enhanced under treatment in EMT6/BRCA1 KO bearing animals.
1 INTRODUCTIONAround 10% of breast cancer cases are attributed to genetic disorders like mutations in BRCA-1/2 genes.Targeted therapy of BRCA-deficient cancers has been achieved using poly(ADP-ribose) polymerase (PARP)inhibitors, which block BRCA-independent DNA repair. To study the effect of this common mutation on tumorbiology in general and specifically the immunological landscape of the tumor microenvironment, we knockedout the BRCA1 gene in the murine EMT6 breast cancer cell line. Subsequently, we analyzed the tumorinfiltrating lymphocyte (TIL) population and the cytokine profiles in serum of tumor bearing animals.
3 CONCLUSIONFurther mechanistic studies as well as comparative studies will elucidate the tumor biology behind these observations and might lead tobeneficial combination strategies in patients suffering from triple negative and BRCA1-/- breast cancer
Figure 1. Mode of action of PARP (A) and checkpoint (B) inhibitors and treatmentregimen in combination (C)
Fig 2. Creation of homozygous EMT6 BRCA1 KO line. Generation of a Brca1 knock-out inmouse breast cancer cell line EMT6 mice based on CRISPR technology using the HDRpathway
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Figure 3. Basic tumor biology characteristics. The volume of individualtumors was determined twice weekly from the day of implantation on. TIL andcytokine analysis was performed at a tumor volume of 1500 mm³.
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B
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Mittica G. et al: Int J Mol Sci. 2016 Jul 20;17(7). pii: E1169. doi: 10.3390/ijms17071169.
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Figure 5. Sensitivity towards PARP and checkpoint inhibitors in combination therapy.Tumor volume over time
Figure 4. Sensitivity towards PARP and checkpoint inhibitors in monotherapy.
monotherapy. The EMT6 model was sensitive towards both checkpoint inhibitor treatments. Combination therapy was more effective in all tested settings. However, Rucaparib + anti CTLA
prolongation of the life span of the treated animals.
volume over time
Figure 6. Sensitivity towards PARP and checkpoint inhibitors in combination therapy.Overall survival
Figure 7. Sensitivity towards PARP and checkpointinhibitors in combination therapy in EMT6 BRCA1 KO (A)and EMT6 (B). TIL analysis
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Figure 8. Sensitivity towards PARP and checkpoint inhibitors in combination therapy in EMT6 BRCA1KO (A) and EMT6 (B). Cytokine profile
A B
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