XPO1 Inhibitor Research Articles

Abstract 738: Targeting metabolic vulnerabilities of endocrine resistant breast cancers using XPO1 and NAMPT inhibition

Abstract Introduction: Approximately 70% of human breast cancers express estrogen receptor alpha (ERα), providing the potential for targeted endocrine therapy for patients. Unfortunately, 30-40% of ER+ patients still experience recurrence and metastasis with a 5-year relative overall survival rate of just 24%. Previously, we described metabolic changes in metastatic breast cancer cells that are driven by single therapies and the metastatic organ microenvironment. Combining selinexor (SEL), a small molecule inhibitor of exportin 1 (XPO1) approved by the FDA for treatment of some hematologic malignancies, with the current therapies used for advanced breast cancers (e.g. tamoxifen) decreased metabolic adaptations in response to individual drug treatments and prevented colony formation in hydrogels and tumor growth in xenograft models. Since we observed a reliance on NAD+ metabolism in cancer cells that are treated with single therapies, in the current study we investigated co-targeting metabolic alterations using SEL and the NAMPT inhibitor KPT-9274. Methods: We established 3D cell culture systems in novel hydrogels derived from decellularized bone, lung, and liver tissues - the major sites of metastasis of ER+ breast cancers, and compared with standard Matrigel culture. Endocrine responsive (MCF7 and T47D) and resistant (MCF7 and T47D cells with ESR1 Y537S or D538G mutations) breast cancer cell lines were treated with SEL or KPT-9274. Tumorigenicity was assessed using in vitro cell viability and colony formation assays and in vivo breast cancer cell line xenografts in athymic mice. Metabolic changes were assessed using whole metabolite profiling by GC-MS and a Seahorse XF analyzer. Results: Combining an FDA-approved XPO1 small molecule inhibitor, Selinexor (SEL) with the NAMPT inhibitors decreased metabolic adaptations in response to individual drug treatments, and prevented colony formation in hydrogels and tumor growth in xenograft models. Conclusions: Targeting drug and metastatic-site induced adaptations to regenerate new vulnerabilities in endocrine-resistant breast tumors are novel. Given the need for better strategies for improving therapy response of metastatic ER+ tumors, our findings show uncovering the role XPO1-NAMPT crosstalk plays in metastatic breast cancer could lead to new combined therapies that reduce mortality. Citation Format: Qianying Zuo, Ayca Mogol, Ozan Imir, Yosef Landesman, Zeynep Madak Erdogan. Targeting metabolic vulnerabilities of endocrine resistant breast cancers using XPO1 and NAMPT inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 738.

Abstract 1380: Synergistic effect of the combination of XPO1 and mTORC1/2 inhibition for the treatment of triple-hit DLBCL

Abstract Background Double/triple-hit lymphoma is an aggressive form of DLBCL with particularly poor outcome. XPO1 (exportin 1) is a well characterized nuclear export protein which is overexpressed in multiple tumor types. XPO1 exports many tumor-suppressor proteins and thus acts as a protooncogene by removing tumor suppressor protein and growth regulatory factors from the nucleus, where they are active, to the cytoplasm. First-in-class, single agent oral XPO1 inhibitor, Selinexor (ATG-010), was recently approved for the treatment of patients with DLBCL (de novo or transformed from follicular NHL) after at least two prior therapies. mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2) are critical mediators of the PI3K-AKT pathway. Signaling by the PI3K/AKT/mTOR pathway is frequently deregulated in NHL. Inhibition of mTOR has shown preclinical and clinical efficacy in treating DLBCL. This study tested the antitumor effects induced by the combination of the XPO1 inhibitor, Selinexor and the dual mTORC1/2 kinase inhibitor, ATG-008 (Onatasertib) on triple-hit DLBCL cells, DoHH2. Methods Firstly, the in vitro 50% inhibition concentration (IC50) of two compounds was determined in DoHH2 cell line using CellTiter-Glo luminescent cell viability assay. The synergy effect of compound ATG-008 in combination with ATG-010 were evaluated by combination index (CI). The synergy effect is calculated by the Chou-Talalay Method [1]. The CI score ranging from 0.1-0.9 suggests very strong to slight synergism. The in vivo combination of the drugs were tested in DoHH2 CDX mouse model. The tumor bearing mouse were treated with vehicle control, Selinexor (5mg/kg, MWF), ATG-008 (10mg/kg, QD) or the combination for 21 days. The tumor size was measured twice a week and tumor growth inhibition (TGI) was evaluated compared with vehicle control group. Results Potent in vitro and in vivo anti-tumor efficacy and synergy has been observed for the combination of the two drugs. The in vitro IC50 for DOHH-2 cell line were 0.85µM and 0.08µM for ATG-008 and Selinexor, respectively. Synergy has been observed for most combo concentrations tested with the strongest synergism observed for ATG-008 (1.7µM) + Selinexor (0.16µM). The combination index was 0.48. In the DoHH2 CDX in vivo study, the mono therapy of ATG-008 showed 41% TGI at day 19 after grouping (p value<0.001), and 19% TGI for Selinexor (p value= 0.147) mono therapy treatment group. The combination of ATG-008 and Selinexor showed 75% TGI (p value<0.01). Conclusions Strong synergism has been observed for the combination of XPO1 and mTORC1/2 inhibition by Selinexor and ATG-008, respectively, suggesting promising therapeutic strategies for double/triple-hit lymphoma patients that warrants further investigation. [1]Chou TC. Cancer Res. 2010 Jan 15;70(2):440-6. Citation Format: Bing Hou, Aihua Wang, Bo Shan, Jay Mei. Synergistic effect of the combination of XPO1 and mTORC1/2 inhibition for the treatment of triple-hit DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1380.

Abstract 1341: Inhibiting the nuclear exporter XPO1 and the antiapoptotic factor BCL2 is synergistic in XPO1 and SF3B1 mutant hematologic malignancies

Abstract Mutations in the nuclear exporter XPO1 and the spliceosomal protein SF3B1 are bona fide cancer drivers and occur across a spectrum of hematologic cancers, specifically in chronic lymphocytic leukemia (CLL), myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Selinexor is a potent, XPO1 inhibitor that was recently approved in diffuse large B-cell lymphoma and is being tested in MDS and AML. The BCL2 inhibitor, venetoclax, is approved for use in CLL and has also shown to be effective in myeloid malignancies. XPO1 inhibition exerts its antineoplastic effects by blocking key tumorigenic pathways, such as NFκB, and decreasing the anti-apoptotic protein MCL1. We therefore hypothesized that combining selinexor and venetoclax would have potential synergy and could be used to target hematologic malignancies bearing XPO1 and SF3B1 mutations. We first determined whether XPO1 and SF3B1 mutant cell lines showed differential response to either selinexor or venetoclax monotherapy. Five lymphoma cell lines; 3 diffuse large B-cell lymphoma (SU-DHL-6, SU-DHL-16, FARAGE) and 2 classical Hodgkin lymphoma (L428 and SUP-HD1), were used based on the presence or absence of XPO1 mutations. SUDHL-16 and SUP-HD1 are heterozygous for the XPO1 E571K hotspot mutation while SUDHL-6, FARAGE and SUP-HD1 are wildtype. These cell lines were used to assess sensitivity to Selinexor or Venetoclax using the CellTiter Glo assay. The XPO1 mutant cells showed increased sensitivity to selinexor (IC50 = 16-35nM vs 41-231nM). Additionally, the XPO1 mutant cell lines showed increased sensitivity to single-agent venetoclax (XPO1 mutant IC50 = 2-13nM vs 5-2853nM). SF3B1 K666N mutant K562 and Nalm-6 cells also showed increased sensitivity to selinexor compared to wildtype cells (IC50 =116-190nM vs 212-1405nM), as well as to venetoclax as compared to wildtype (IC50 =149nM-1.28 μM vs 1.26-1.4μM). Next, we tested the synergy of the combination of these drugs in the XPO1 and SF3B1 mutant cell lines. Increasing concentrations of the individual drugs were applied to each individual cell line in a 6x6 matrix and the Bliss Independence model was used to calculate synergy of the combination. As hypothesized, the combination of Selinexor and venetoclax showed synergy in both the XPO1 and SF3B1 mutant cell lines (synergy scores 4.5-16.5). Western blots of treated wildtype versus mutant cells showed decreased levels of p65 and MCL1 with selinexor (but not venetoclax) that was enhanced with combination therapy, specifically in mutant cell lines. In conclusion, inhibiting the nuclear exporter XPO1 and the anti-apoptotic factor BCL2 is synergistic in both XPO1 and SF3B1 mutant cells. This combination is highly promising as an all oral regimen for hematologic malignancies bearing these mutations. Further, preclinical testing in in vivo using XPO1 and SF3B1 mutant and wildtype mice is ongoing. Citation Format: Tulasigeri M. Totiger, Sana Chaudhry, Jumana Afaghani, Justin Taylor. Inhibiting the nuclear exporter XPO1 and the antiapoptotic factor BCL2 is synergistic in XPO1 and SF3B1 mutant hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1341.

Abstract 3040: Inhibition of Exportin-1 as part of combinatorial treatment for patients with high-risk neuroblastoma

Abstract Background: In many adult cancers, upregulation of the nuclear-to-cytoplasmic transport protein Exportin-1 (XPO1) correlates with poor outcome and responsiveness to selinexor, an FDA-approved XPO1 inhibitor. Similar data are emerging in childhood cancers, for which selinexor is being evaluated in early phase clinical studies. Our aims are to identify patients most likely to benefit from selinexor combination therapy and define the cellular mechanism responsible this effect. We hypothesize that selinexor and the proteasome inhibitor, bortezomib, act synergistically to decrease NF-kB-mediated proliferation by halting excessive exportation and proteasome degradation of IkB, an XPO1 cargo protein. Methods: We performed ion intensity-based label-free semiquantitative proteomic profiling on primary tumor material from fifty patients with uniformly treated high-risk NB, half of whom were long term survivors and half of whom died <18 months from diagnosis from disease progression. We cross-referenced this dataset with gene expression profiling data available through the R2 database. A panel of neuroblastoma cell lines was screened for XPO1 expression using Western blotting, sensitivity to selinexor and bortezomib as single agents, and in combination using cellular viability assays. Localization and quantification of IkB after vehicle or selinexor treatment was done by confocal imaging, using immunofluorescent labeling and Volocity software. Flow cytometry was used to define treatment effect on cell cycle arrest and apoptosis using BrdU and Annexin-V labeling, respectively. siRNA-mediated knockdown of IkB was used to investigate the relationship between presence of IkB and drug effectiveness. NF-kB activity was measured with a luciferase assay. Results: High tumor protein and gene expression of XPO1 correlates with poor outcome. Neuroblastoma cell lines demonstrate variably high XPO1 expression compared to normal tissue, as well as sensitivity to selinexor (IC50 range 4-300nM). IkB is retained in the nuclear compartment after treatment with selinexor. When selinexor is used in combination with bortezomib the effect on cell proliferation is synergistic. Flow cytometry demonstrates that selinexor alone contributes to G1-0 arrest while combination treatment results in significantly enhanced apoptosis. Measurement of NF-kB transcriptional activity shows decrease in activity with single agents and the least amount of activity in combination and siRNA against IkB diminishes the effect of single and combination treatment. Conclusions: This work demonstrates that XPO1 expression is a biomarker of highly aggressive neuroblastoma and may define patients most likely to be responsive to treatment with selinexor-based combinatorial therapies. Synergistic treatment effect with selinexor and bortezomib is mediated, in part, through the presence of IkB to regulate NF-kB signaling. Citation Format: Basia Galinski, Yossef Landesman, Daniel Weiser. Inhibition of Exportin-1 as part of combinatorial treatment for patients with high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3040.

AML-Related NPM Mutations Drive p53 Delocalization into the Cytoplasm with Possible Impact on p53-Dependent Stress Response.

Simple SummaryNucleophosmin (NPM) is one of the most abundant nucleolar proteins and its mutations frequently occur in acute myeloid leukemia (AML). The mutations cause aberrant cytoplasmic localization of mutated protein (NPMmut) and often mediate dislocation of NPM interaction partners. Tumor suppressor p53 is known to interact with NPM in response to genotoxic stress and its cytoplasmic localization is an unfavorable prognostic factor in cancers. This study aims to characterize the NPM-p53 interaction and to elucidate the effect of the NPM mutations on p53 localization and expression in live cells. In addition, the cellular dynamics of NPMmut and p53 after treatment with nuclear export inhibitor Selinexor is described and the mechanism of the Selinexor action proposed. Our results contribute to a better understanding of the oncogenic potential of NPM mutations.Nucleophosmin (NPM) interaction with tumor suppressor p53 is a part of a complex interaction network and considerably affects cellular stress response. The impact of NPM1 mutations on its interaction with p53 has not been investigated yet, although consequences of NPMmut-induced p53 export to the cytoplasm are important for understanding the oncogenic potential of these mutations. We investigated p53-NPM interaction in live HEK-293T cells by FLIM-FRET and in cell lysates by immunoprecipitation. eGFP lifetime-photoconversion was used to follow redistribution dynamics of NPMmut and p53 in Selinexor-treated cells. We confirmed the p53-NPMwt interaction in intact cells and newly documented that this interaction is not compromised by the NPM mutation causing displacement of p53 to the cytoplasm. Moreover, the interaction was not abolished for non-oligomerizing NPM variants with truncated oligomerization domain, suggesting that oligomerization is not essential for interaction of NPM forms with p53. Inhibition of the nuclear exporter XPO1 by Selinexor caused expected nuclear relocalization of both NPMmut and p53. However, significantly different return rates of these proteins indicate nontrivial mechanism of p53 and NPMmut cellular trafficking. The altered p53 regulation in cells expressing NPMmut offers improved understanding to help investigational strategies targeting these mutations.

XPO1 Inhibition Targets Transcriptional Vulnerability of FLT3-ITD+D835 Double Mutant AML through p53 Accumulation and Inhibition of Oncogenic Transcription Factors: Lesson Learned from Cage Sequencing of Primary AML Cells

Acute myeloid leukemia (AML) with Fms-like tyrosine kinase 3 / the internal tandem duplications (FLT3 /ITD mutations) present a major clinical challenge, because of short remission duration and higher relapse rates. FLT3-targeted therapies using tyrosine kinase inhibitors (TKIs) often promote acquisition of additional point mutations in the tyrosine kinase domains (TKD) mutations, most commonly at D835 within the activation loop.To elucidate the alterations of transcriptome signatures of FLT3 /ITD-D835 mutants in AML, we performed cap analysis of gene expression (CAGE) sequencing of 14 AML patient samples with FLT3 /ITD and 12 patient samples with FLT3 /ITD-D835 mutations. CAGE identifies and quantifies the 5' ends of capped mRNA transcripts, which enables the identification of transcription start sites (TSS) necessary for gene expression. The TSS of genes altered in AML samples with FLT3 /ITD-D835 compared with FLT3 -ITD were mapped. CAGE detected upregulation of 378 and downregulation of 77 genes, respectively (false discovery rate; FDR < 0.05). Ingenuity Pathway Analysis (IPA) highlighted the activation of transcription factors NFKBIA (IκBα), RELA (NFκB subunit), HIF1α, EP300 (co-activator of HIF1α), TP73, PRDM1 (regulator of TP53 activity), NUPR1 (regulator of p21 relocalization), and FOXO3 (post-transcriptional regulators of MYC) in FLT3 /ITD-D835 mutant primary patient samples compared to FLT3 /ITD primary samples (Z score >1.9).To validate these transcriptional changes, we utilized paired isogenic FLT3 /ITD or FLT3 /ITD-D835Y transfected Ba/F3 cell lines. CAGE detected upregulation of 523 and downregulation of 373 genes, respectively, in FLT3 /ITD-D835 compared to FLT3 /ITD cells (FDR < 0.05). IPA indicated activation of HIF1α and MYC and inhibition of TP53 in FLT3 /ITD-D835 compared to FLT3 /ITD cells. Immunoblot analysis confirmed higher expression of c-Myc, IκBα and HIF1α and lower expression of TP53 in FLT3 /ITD-D835 compared to FLT3 /ITD cells. In turn, FLT3 downstream targets phospho- (p-) ERK, p-AKT, and p-STAT5 were significantly lower in FLT3 /ITD-D835 than in FLT3 /ITD cells. These results indicate that secondary FLT3 -D835 mutations activate oncogenic transcription factors c-Myc, IκBα and HIF1α, inhibit tumor suppressor TP53, and downregulate FLT3 downstream signaling pathways ERK, AKT, and STAT in FLT3 /ITD mutated AML cells.Exportin 1 (XPO1) mediates the nucleo-cytoplasmic transport, and is overexpressed in AML cells with FLT3 /ITD mutations (Kojima, Blood, 2013) . TP53 and IκBα are XPO1 cargos, and XPO1 inhibitors are known to upregulate TP53 and block c-MYC and NFκB signaling. We therefore examined the anti-leukemia activity of clinically available XPO1 inhibitor selinexor in FLT3 /ITD and FLT3 /ITD-D835 Ba/F3 cells. As expected, selinexor effectively inhibited cell proliferation in both FLT3 /ITD and FLT3 /ITD-D835 mutated cells at IC50 of 0.3 μM and 0.1 μM, respectively (48 hrs). CAGE detected upregulation of 1,590 and 2,220 genes and downregulation of 1,238 and 2,640 genes, respectively by selinexor treatment in FLT3 /ITD and FLT3 /ITD-D835 Ba/F3 cells, (FDR < 0.05). IPA analysis highlighted TP53, NUPR1 and CDKN2A (ARF; stabilizer of TP53) as the top three activated transcription factors by selinexor both in FLT3 /ITD and FLT3 /ITD-D835 cells. Immunoblot assay detected that selinexor significantly upregulated TP53, and downregulated IκBα and HIF1α both in FLT3 /ITD and FLT3 /ITD-D835 Ba/F3 cells. Selinexor treatment suppressed c-Myc and increased cleaved caspase 3 more prominently in FLT3 /ITD-D835 cells, but also upregulated p-ERK, p-AKT, p-STAT5 and p-STAT3, more notable in FLT3 /ITD cells. MCL1 and mTOR downstream p-4EBP1 and p-pS6 were repressed by selinexor in FLT3 /ITD-D835 but not in FLT3 /ITD cells.Collectively, the primary mechanism underlying the sensitivity to XPO1 inhibition of FLT3 /ITD-D835 cells is intolerance to the accumulation of nuclear TP53 and inhibition of multiple oncogenic transcription factors, including c-Myc, IκBα and HIF1α, which confer pro-survival activity independent of FLT3 downstream signaling. These findings indicate that XPO1 (CRM1) inhibition is a promising therapeutic strategy for AML patients with FLT3 /ITD and secondary acquired D835 mutations. DisclosuresHarada:Celgene: Research Funding; NIPPON SHINYAKU CO.: Speakers Bureau; NOVARTIS: Research Funding. Andreeff:Daiichi Sankyo: Consultancy.

Exportin-1 Connects Dynamic Transcription and Translation of Genotoxic Stress Genes in Diffuse Large B-Cell Lymphoma Patients

Exportin-1 (XPO1), that exports a subset of proteins from the nucleus to the cytoplasm, is amplified in 30% of DLBCL. Regardless of XPO1 gene status, DLBCLs require XPO1 to proliferate and survive as demonstrated in pre-clinical (Marullo et al. AACR 2015) and clinical (Kuruvilla at al. Blood 2017) studies with the covalent XPO1 inhibitor selinexor. However, the mechanism underlying such dependency remains unknown.We analyzed the expression of XPO1 protein in a cohort of 57 DLBCL patients by IHC and correlated with XPO1 gene status and clinical outcome. XPO1 increased copy number is associated with elevated protein expression (p = 0.03) and, importantly, patients with elevated XPO1 levels (XPO1 high) are less likely to respond to RCHOP as refractory/early relapse diseases (p = 0.005). This suggests that XPO1 high DLBCL could be biologically fitter to tolerate genotoxic stress. To explore this hypothesis, we compared gene expression profiles (RNA-sequencing) of XPO1 high (n = 15) vs. XPO1 low (n = 16) DLBCLs by DESeq2. XPO1 high DLBCLs were enriched (q<0.005) for genes regulating “cell proliferation”, “DNA damage response and repair”, “mRNA processing” and “RNA export”; whereas depleted (q<0.05) for genes regulating “immune response”. Gene Set Enrichment Analysis also revealed that XPO1 high were enriched for transcripts up-regulated in highly aneuploid tumors (FDR = 0, NES = 2.19). All these findings were validated in an independent cohort of 72 DLBCL patients.To gain insight into the mechanism by which XPO1 enhances genotoxic stress tolerance, we exposed a panel of 5 DLBCL cell lines to DNA damaging agents (etoposide, doxorubicin, mechloretamine) with or without XPO1 inhibition (selinexor). Comet assay experiments revealed that selinexor did not affect the amount of DNA damage induced by chemotherapy, but delayed cell's ability to repair the DNA damage (P>0.005). This translated into a significant increase of sensitivity toward otherwise sub-lethal effect of chemotherapy, as all the combinations tested showed synergistic effect on growth inhibition. To determine the existence of a therapeutic window to capitalize on this effect, we evaluated the combination of CHOP +/- selinexor in an XPO1 high DLBCL patient-derived xenograft mice model (n = 40). Combination treatment achieved higher anti-lymphoma effect compared to either selinexor or CHOP alone (p>0.005 and p= 0.0015, respectively). Importantly, no increase in toxicity was observed by biochemistry or pathological examination. Thus, proliferating DLBCL cells but not normal cells rely on XPO1 to survive exogenous genotoxic stress.To characterize the molecular consequences of XPO1 inhibition, we quantified the expression of proteins involved in DNA damage response and repair (immunoblotting). Exposure to selinexor decreased the expression of a subset of these proteins including CHEK1, BRCA1, BCL6 and RAD51. This reduction was neither due to decreased gene transcription (qRT-PCR) nor to increased protein degradation (cycloheximide chase). Instead, analysis of nascent proteins by L-azido-homo-alanine labeling revealed that XPO1 inhibition decreases the synthesis of specific DNA damage repair proteins whose increased turnover is required to survive genotoxic stress. Since XPO1 inhibition affected only a subset of proteins, the mechanism may involve selective decrease in nuclear export of mRNA templates. Thus, we quantified the nuclear/cytoplasmic ratio of mRNAs encoding for DNA damage repair proteins in DLBCL cells treated with selinexor. XPO1 inhibition resulted in nuclear retention of BRCA1, RAD51, BCL6 and CHEK1 mRNAs but not of β-actin. Since XPO1 is unable to directly bind mRNAs, we reasoned that its inhibition may impair the export of proteins carrying mRNA (ribonucleoproteins). Indeed, we identified eIF4E as one of these cargoes. Immunofluorescence experiments revealed that eIF4E is retained in the nucleus of DLBCL cells upon XPO1 inhibition and its nuclear retention is enhanced in etoposide-treated cells. Moreover, RIP-qPCR experiments revealed that eIF4E binds several DNA damage repair mRNAs such as CHEK1, BCL6 and BRCA1 in the nucleus of DLBCL cells and this binding increases significantly in response to DNA damage.Overall, our data demonstrate that XPO1, by regulating the selective export of mRNA-binding proteins, promotes genotoxic stress tolerance thus conferring a survival advantage to XPO1high DLBCLs. DisclosuresYang:Regeneron Pharmaceuticals: Employment. Cerchietti:Leukemia and Lymphoma Society: Research Funding; Lymphoma Research Foundation: Research Funding; Celgene: Research Funding; Weill Cornell Medicine - New York Presbyterian Hospital: Employment.

Inhibition of XPO1 By KPT-330 (Selinexor) Enhances Cell Death Induced By the Bcl-2 Selective Inhibitor ABT-199 (Venetoclax) through Downregulation of Mcl-1 in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a serious disease and despite an improved understanding of the biology of AML, little has changed in the treatment of the disease over the last four decades. The 5 year survival rates of AML remain frustratingly low. Resistance to frontline chemotherapy remains the main cause of treatment failure; therefore a new treatment modality is urgently needed.Bcl-2 is a promising therapeutic target; it is overexpressed in both bulk AML cells and leukemia-initiating cells. The Bcl-2-selective inhibitor ABT-199 (Venetoclax) shows promising anti-leukemic activity in patients with high-risk relapsed/refractory AML or unfit for intensive chemotherapy. However, resistance through another Bcl-2 family protein, Mcl-1, was shown by our lab and others to limit the anti-leukemic activity of ABT-199. Previous studies from our lab have shown that ABT-199 monotherapy decreases the association of Bim with Bcl-2 but is compensated for by its increased association with Mcl-1. We also found that ABT-199 increases cellular Mcl-1 levels by extending the protein's half-life. Based on these previous studies we tested the efficacy of direct inhibition of Bcl-2 by ABT-199 and Mcl-1 by A-1210477. ABT-199 and A-1210477 synergistically induced apoptosis in both ABT-199-resistant and sensitive AML cells. This combination treatment disrupted binding of Bim with Bcl-2 and Mcl-1 concurrently, with no observed binding of Bim to Bcl-xL. This work has demonstrated that targeting both Bcl-2 and Mcl-1 is necessary for AML treatment. However, our work also indicated that direct targeting of Mcl-1 with A-1210477 to enhance the anti-leukemic activity of ABT-199 might cause increased toxicity. Thus, indirect targeting of Mcl-1 may represent a better approach to enhance the anti-leukemic activity of ABT-199 in AML.It was recently reported that targeting exportin 1 (XPO1), a nuclear exporter overexpressed in AML cells, selectively targets leukemia-initiating cells and can decrease Mcl-1 levels in cancer cells, though the underlying mechanism is unknown. KPT-330 (Selinexor) is a XPO1-selective inhibitor which is currently being tested in clinical trials for AML. Preliminary results have shown that KPT-330 induces a response at well-tolerated doses in AML and other hematological malignancies. Thus, XPO1 inhibition may represent a viable strategy for downregulating Mcl-1 to synergize with ABT-199 treatment in AML.To test our hypothesis we tested various concentrations of ABT-199 and KPT-330 alone and in combination in AML cell lines and primary patient samples. At 24 h, synergistic induction of apoptosis was observed between the two drugs in THP-1, CTS, Molm-13, and OCI-AML3 AML cell lines. Synergy was also found in 4 relapsed and 6 newly diagnosed primary AML patient samples. To test the effect of drug treatment on leukemia-initiating cells, we performed colony formation assays on primary AML patient samples post drug treatment and found that combination treatment significantly enhanced inhibition of colony formation when compared to single drug treatment, demonstrating that KPT-330 combined with ABT-199 is effective in targeting the leukemic bulk and the leukemia-initiating cells.To determine the mechanism by which the combination works, we measured the protein levels of various Bcl-2 family members. Decreased protein levels for Mcl-1 and surprisingly Bim were observed post KPT-330 or KPT-330 plus ABT-199 treatment, while protein levels for Bcl-2 and Bcl-xL remained unchanged. A time course experiment revealed that Mcl-1 levels decreased at apoptosis initiation while Bim levels decreased after apoptosis initiation, suggesting that Mcl-1 plays a critical role in apoptosis induced by the drug combination, while Bim may not. Mcl-1 knockdown enhanced combination treatment of ABT-199 and KPT-330. Reciprocal Mcl-1 overexpression partially rescued the cells from drug treatment but was incomplete, likely due to Mcl-1 overexpression not being high enough and/or non-Mcl-1 pathways through which KPT-330 acts. In summary, Mcl-1 is modulated by KPT-330 treatment alone and in combination with ABT-199 and this modulation of Mcl-1 plays an important role in initiation of apoptosis in response to treatment. Additional studies are underway to further elucidate the molecular mechanisms of combination ABT-199 and KPT-330 treatment and to determine the in vivo anti-leukemic efficacy in NSG mouse AML models. DisclosuresNo relevant conflicts of interest to declare.

Xpo-1 Inhibition Synergizes with BCR Inhibition, Blocks Tumor Growth and Tumor-Associated Macrophages Infiltration and Prolongs Survival in a Bioluminescent Animal Model of Primary Central Nervous System Lymphoma

Primary central nervous system lymphoma (PCNSL) is an infrequent and aggressive non-Hodgkin lymphoma usually classified as diffuse large B-cell lymphoma (DLBCL), being most of the cases more closely related to activated B-cell type (ABC). PCNSL is associated with dismal prognosis, due in part to the difficulty for many chemotherapeuticals to cross the blood brain barrier.Selinexor (KPT-330) is a Selective Inhibitor of Nuclear Export (SINE) compound that binds to and inactivates XPO-1 protein and induces anti-tumor effects mainly due to forced nuclear retention and activation of tumor suppressor proteins. Selinexor, that has shown excellent brain penetrance and promising results in pre-clinical models of glioblastoma, can inhibit both BCR and NF-kB signaling in malignant B-cells.In order to provide a pre-clinical rationale for the design of new therapeutic strategies in the PCNSL, we assessed the role of XPO-1 inhibition using intracerebral xenograft murine models.To compare the sensitivity of DLBCL-derived cell lines to selinexor, we determined the inhibitory concentration 50 (IC50) in terms of survival and proliferation after 96 hours in 4 ABC and 5 GCB DLBCL cell lines. Analysis showed that DLBCL cell lines have equivalent sensitivity to selinexor, regardless the cell of origin. Since SINE compounds have been shown to inhibit BCR signaling, we next tested the potential synergy between ibrutinib and selinexor. In 3 out of 4 ABC-DLBCL cell lines there was a strong synergy. In contrast, none of the 3 GCB-DLBCL cell lines analyzed were sensitive to up to 100 µM single agent ibrutinib; interestingly, however, treatment with selinexor sensitized SUDHL4 (GCB) cells to ibrutinib and the combination index values indicated strong synergism between the two drugs.We then established an orthotopic xenograft model of PCNSL by stereotactic injection of OCI-Ly10 cells stably transfected with luciferase into the brain parenchyma of nude athymic mice. Eleven days after the injection of cells all animals had developed detectable tumors confined to the CNS. Tumor size was measured and animals were randomly distributed into drug or vehicle experimental group. At this time point mice were treated with 5mg/kg of selinexor or vehicle via oral gavage three times a week; subsequently, bioluminiscence was assessed twice a week. Treatment with selinexor significantly blocked tumoral growth (two-way ANOVA: p< 0.0001; figure 1A). Specific time-point analysis showed that differences were significant as soon as 12 days after treatment (2,255e+008 +/- 8,648e+007 vs 8,670e+006 +/- 1,981e+007). Also, treatment with selinexor increased mice survival, with a median survival of 48 days in the treatment group compared to 34 days in the vehicle group (p< 0.0001; figure 1B). At final point, histopathological analysis showed diffuse infiltration in meninges and cerebral parenquima of highly proliferative CD20-positive B-cells (virtually 100% Ki-67-positive malignant B cells). Since the infiltration of tumoral associated macrophages (TAMs) has been related to worse prognosis in different lymphomas, including PCNSL, we analyzed the infiltration by F4/80+ TAMs after two weeks of treatment with selinexor or vehicle. We observed that all tumors from vehicle-treated mice were infiltrated with TAMs, whereas treatment with selinexor significantly reduced the presence of TAMs as assessed by immunohistochemistry, especially in the parenquima. Next, we evaluated the potential synergy between ibrutinib and selinexor in vivo. For that 12 mice with equivalent tumor size were assigned to each of the following four groups: selinexor only (5mg/kg twice a week), ibrutinib only (25mg/kg daily), combination or vehicle. The combination of both drugs significantly increased survival compared to both treatments as single agent, whereas there was no significant difference between ibrutinib and selinexor treatments alone (figure 1C). Tumoral growth was equally blocked by all treatments (figure 1D). Currently, we are evaluating the effect of the combination in the infiltration and immunophenotype of TAMs, including M1 vs M2 phenotype and PD1 expression. Results will be available at the time of the meeting.Selinexor proved to be an active agent in blocking tumoral growth in DLBCL infiltrating CNS, this providing pre-clinical evidence for the development of selinexor as new therapeutic option for PCNSL or DLBCL with CNS involvement. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Phase I/II Study of Liposomal Doxorubicin (DOX) in Combination with Selinexor (SEL) and Dexamethasone (Dex) for Relapsed and Refractory Multiple Myeloma (RRMM)

Background: SEL, an oral first-in-class inhibitor of XPO1 showed activity in clinical trials for hematologic malignancies. SEL inhibits DNA damage repair and exhibits marked synergy with doxorubicin in preclinical myeloma models. SEL/Dex results in a 20% response rate in penta-refractory myeloma patients. We report here the results of a phase I/II trial of SEL in combination with DOX and Dex in patients with RRMM.Methods: Eligible patients had RRMM and received ≥ 2 prior therapies including lenalidomide and a proteasome inhibitor. Treatment consisted of a loading phase with SEL and Dex for 1-2 weeks; an induction phase with DOX 20 mg/m2 IV D1, SEL and Dex (once weekly) and a maintenance phase of weekly SEL/Dex. Two loading phases were evaluated: A. SEL/Dex twice weekly for 2 weeks, B. One dose of SEL/Dex (table 1). Primary and secondary end points: maximum tolerated dose (MTD) / recommended phase II dose (RP2D) of the combination, and overall response rate (ORR) per International Myeloma Working Group (IMWG) criteria.Results: Twenty-seven patients were enrolled (median age of 62 years, median of 6 prior lines (range 2-10)). No dose limiting toxicities (DLT) were noted in dose level (DL) 1. Two patients experienced a DLT in DL2 (Gr4 thrombocytopenia and Gr 3 nausea). The loading phase was shortened to 1 dose of SEL (80mg) on day -7 (DL2m). 1/6 patients experienced a DLT on DL2m (Gr3 hyponatremia). Two patients experienced a DLT in dose level 3m (Grade 3 ALT and Grade 3 confusion). The recommended phase II dose was determined to be DL2m (SEL 80mg D1,8,15, DOX 20mg/m2 D1 and Dex 40 mg D1,8,15). Three patients died on study (1 from RSV pneumonia, one from pneumocystis Jiroveci and 1 from advanced myeloma). The most common Grade 3/4 at least possibly related adverse events are as follows: thrombocytopenia 33%, neutropenia 33%, asymptomatic hyponatremia 30%, anemia 26%, nausea / vomiting 11%, hyperglycemia 11%, diarrhea 7%, and fatigue 7% (table 2). Of the 14 patients treated at the RP2D, one was not evaluable (NE), one had a PR and 1 a MR at the time of this analysis. Per protocol, this current level of response does not justify proceeding with the second stage of the phase II study. Of the 27 total patients treated, we noted 2 VGPR, 2 PR, 3MR, 8SD, 4NE with an ORR of 15%, clinical benefit rate (MR and better) of 26% by intent to treat. There was increased nuclear localization of Ikβ post selinexor treatment in responders compared to non-responders (Nuclear / cytoplasmic ratio mean 1.15 ±0.06 versus 0.93 ±0.09; p=0.04).Conclusions: The addition of DOX to SEL/Dex, while reasonableywell tolerated, does not appear to improve the ORR noted with SEL/Dex in this heavily pretreated patient population. [Display omitted] DisclosuresBaz:BMS: Research Funding; celgene: Honoraria, Research Funding; karyopharm: Research Funding; takeda: Research Funding; merck: Research Funding; Sanofi: Research Funding. Zonder:Janssen: Consultancy; BMS: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Takeda: Consultancy; Pharmacyclics: Other: Data Safety Monitoring Committee; Prothena: Consultancy.

PSEN1-selective gamma-secretase inhibition in combination with kinase or XPO-1 inhibitors effectively targets T cell acute lymphoblastic leukemia

BackgroundT cell acute lymphoblastic leukemia (T-ALL) is a high-risk subtype that comprises 10–15% of childhood and 20–25% of adult ALL cases. Over 70% of T-ALL patients harbor activating mutations in the NOTCH1 signaling pathway and are predicted to be sensitive to gamma-secretase inhibitors. We have recently demonstrated that selective inhibition of PSEN1-containing gamma-secretase complexes can overcome the dose-limiting toxicity associated with broad gamma-secretase inhibitors. In this study, we developed combination treatment strategies with the PSEN1-selective gamma-secretase inhibitor MRK-560 and other targeted agents (kinase inhibitors ruxolitinib and imatinib; XPO-1 inhibitor KPT-8602/eltanexor) for the treatment of T-ALL.MethodsWe treated T-ALL cell lines in vitro and T-ALL patient-derived xenograft (PDX) models in vivo with MRK-560 alone or in combination with other targeted inhibitors (ruxolitinib, imatinib or KPT-8602/eltanexor). We determined effects on proliferation of the cell lines and leukemia development and survival in the PDX models.ResultsAll NOTCH1-signaling-dependent T-ALL cell lines were sensitive to MRK-560 and its combination with ruxolitinib or imatinib in JAK1- or ABL1-dependent cell lines synergistically inhibited leukemia proliferation. We also observed strong synergy between MRK-560 and KPT-8602 (eltanexor) in all NOTCH1-dependent T-ALL cell lines. Such synergy was also observed in vivo in a variety of T-ALL PDX models with NOTCH1 or FBXW7 mutations. Combination treatment significantly reduced leukemic infiltration in vivo and resulted in a survival benefit when compared to single treatment groups. We did not observe weight loss or goblet cell hyperplasia in single drug or combination treated mice when compared to control.ConclusionsThese data demonstrate that the antileukemic effect of PSEN1-selective gamma-secretase inhibition can be synergistically enhanced by the addition of other targeted inhibitors. The combination of MRK-560 with KPT-8602 is a highly effective treatment combination, which circumvents the need for the identification of additional mutations and provides a clear survival benefit in vivo. These promising preclinical data warrant further development of combination treatment strategies for T-ALL based on PSEN1-selective gamma-secretase inhibition.

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Covering the Cover

Selinexor, a novel selective inhibitor of nuclear export, reduces SARS-CoV-2 infection and protects the respiratory system in vivo

The novel coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the recent global pandemic. The nuclear export protein (XPO1) has a direct role in the export of SARS-CoV proteins including ORF3b, ORF9b, and nucleocapsid. Inhibition of XPO1 induces anti-inflammatory, anti-viral, and antioxidant pathways. Selinexor is an FDA-approved XPO1 inhibitor. Through bioinformatics analysis, we predicted nuclear export sequences in the ACE-2 protein and confirmed by in vitro testing that inhibition of XPO1 with selinexor induces nuclear localization of ACE-2. Administration of selinexor inhibited viral infection prophylactically as well as therapeutically in vitro. In a ferret model of COVID-19, selinexor treatment reduced viral load in the lungs and protected against tissue damage in the nasal turbinates and lungs in vivo. Our studies demonstrated that selinexor downregulated the pro-inflammatory cytokines IL-1β, IL-6, IL-10, IFN-γ, TNF-α, and GMCSF, commonly associated with the cytokine storm observed in COVID-19 patients. Our findings indicate that nuclear export is critical for SARS-CoV-2 infection and for COVID-19 pathology and suggest that inhibition of XPO1 by selinexor could be a viable anti-viral treatment option.

Selected Articles from This Issue

Zotiraciclib, a cyclin-dependent kinase 9 (CDK9) inhibitor, was previously shown to synergize with temozolomide (TMZ) in preclinical studies. To translate this finding to the clinic, Wu and colleagues performed a clinical trial of zotiraciclib plus TMZ in recurrent high-grade astrocytoma. This two-stage phase I trial simultaneously optimized the dosing and schedule of the combination of zotiraciclib and temozolomide using a BOIN dose-finding design. In addition to establishing the optimal dosing schedule for zotiraciclib and temozolomide, in-depth analyses of zotiraciclib-induced neutropenia, PK/p. and longitudinal symptom burden analysis were performed. The comprehensive information from this multifaceted early-stage investigation supports further clinical trials of this agent.Nuclear exporter protein exportin1/XPO1 overexpression in hematological malignancies leads to cytoplasmic mislocalization and functional inactivation of tumor suppressors. Specific inhibitor of nuclear export (SINE) compounds block XPO1-mediated protein transport, retaining tumor suppressors in the nucleus. Seymour and colleagues demonstrate preclinical efficacy and clinical validation of XPO1 inhibitor selinexor in non-Hodgkin lymphoma with a phase I study of R-CHOP plus weekly selinexor as frontline treatment of NHL. This combination was well tolerated, and an MTD was not reached. An ORR of 100% and a CR rate of 90% were observed. Further study of this combination in lymphoma is warranted.While neoadjuvant chemoradiation (nCRT) has improved the survival of patients with resectable esophageal adenocarcinoma (rEAC), nearly half of patients succumb to their disease within 5 years. To assess improving this treatment strategy, van den Ende and colleagues assessed the combination of nCRT with PD-L1 inhibition in patients with rEAC. The pathological complete response rate was 25%, but there was no significant improvement in survival compared with nCRT alone. However, potential biomarkers of response were identified. An IFNy signature was associated with response, while low levels of cytotoxic lymphocytes or the presence of checkpoint inhibition correlated with lack of response. Further study is necessary to clarify the therapeutic potential of this combination in rEAC.IL-15 is an important cytokine for the function of NK cells, and IL-15 activation has been proposed as a strategy for immunotherapy. Foltz and colleagues conducted a clinical trial of N-803, an IL-15 receptor superagonist complex, with rituximab in patients with relapsed/refractory non-Hodgkin's lymphoma. This combination was well tolerated and led to prolonged responses in a subset of patients. N-803 treatment led to the activation of NK cells, CD8+ T cells, and monocytes. Further clinical study of N-803 is needed to identify patient populations for whom this agent will be beneficial, as well as appropriate combination treatments.

Proteomics identify nuclear export as a targetable pathway in neuroblastoma: Comment on “XPO1 inhibition with selinexor synergizes with proteasome inhibition in neuroblastoma by targeting nuclear export of IκB”

Neuroblastoma (NBL) is an embryonal malignancy of childhood with poor outcomes for patient with high-risk disease. Multimodal treatment approaches have improved outcomes but at the cost of significant toxicity, and there is no durable therapeutic approach for relapsed disease. As NBL has no singular oncogenic driver, targeted therapeutic options have been limited. Galinski et al report the results of a proteomic screen of neuroblastomas and identify the nuclear export protein XPO1 as a protein that is preferentially expressed and located in neuroblast nuclei. XPO1 overexpression is associated with nuclear export of IκB and increased NF-κB activity, both of which can be abrogated in NBL cell lines with the XPO1 inhibitor Selinexor with or without the proteasome inhibitor bortezomib. This work highlights new strategies for therapeutic target identification and the novel identification of nuclear export as a targetable oncogenic pathway across malignancies.

MiR-34a-Mediated Survivin Inhibition Improves the Antitumor Activity of Selinexor in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive disease with limited therapeutic options. Here, we pursued a combinatorial therapeutic approach to enhance the activity of selinexor, the first-in-class XPO1 inhibitor, by miR-34a ectopic expression in human TNBC experimental models. Anti-proliferative activity induced by selinexor and miR-34a expression, singly and in combination, was evaluated by MTS assay and cell counting. The effect of treatments on survivin and apoptosis-related proteins was assessed by western blotting and ELISA. The antitumor and toxic effects of individual and combined treatments were evaluated on TNBC orthotopic xenografts in SCID mice. Selinexor consistently showed anti-proliferative activity, although to a variable extent, in the different TNBC cell lines and caused the impairment of survivin expression and intracellular distribution, accompanied by apoptosis induction. Consistent with in vitro data, the XPO1 inhibitor variably affected the growth of TNBC orthotopic xenografts. miR-34a cooperated with selinexor to reduce survivin expression and improved its anti-proliferative activity in TNBC cells. Most importantly, miR-34a expression markedly enhanced selinexor antitumor activity in the less sensitive TNBC xenograft model, in absence of toxicity. Our data form a solid foundation for promoting the use of a miR-34a-based approach to improve the therapeutic efficacy of selinexor in TNBC patients.

XPO1 inhibition with selinexor synergizes with proteasome inhibition in neuroblastoma by targeting nuclear export of IkB

Across many cancer types in adults, upregulation of the nuclear-to-cytoplasmic transport protein Exportin-1 (XPO1) correlates with poor outcome and responsiveness to selinexor, an FDA-approved XPO1 inhibitor. Similar data are emerging in childhood cancers, for which selinexor is being evaluated in early phase clinical studies. Using proteomic profiling of primary tumor material from patients with high-risk neuroblastoma, as well as gene expression profiling from independent cohorts, we have demonstrated that XPO1 overexpression correlates with poor patient prognosis. Neuroblastoma cell lines are also sensitive to selinexor in the low nanomolar range. Based on these findings and knowledge that bortezomib, a proteasome inhibitor, blocks degradation of XPO1 cargo proteins, we hypothesized that combination treatment with selinexor and bortezomib would synergistically inhibit neuroblastoma cellular proliferation. We observed that selinexor promoted nuclear retention of IkB and that bortezomib augmented the ability of selinexor to induce cell-cycle arrest and cell death by apoptosis. This synergy was abrogated through siRNA knockdown of IkB. The synergistic effect of combining selinexor and bortezomib in vitro provides rationale for further investigation of this combination treatment for patients with high-risk neuroblastoma.

Chemoproteomic Profiling of Covalent XPO1 Inhibitors to Assess Target Engagement and Selectivity

Selinexor, a covalent XPO1 inhibitor, is approved in the USA in combination with dexamethasone for penta-refractory multiple myeloma. Additional XPO1 covalent inhibitors are currently in clinical trials for multiple diseases including hematologic malignancies, solid tumor malignancies, glioblastoma multiforme (GBM), and amyotrophic lateral sclerosis (ALS). It is important to measure the target engagement and selectivity of covalent inhibitors to understand the degree of engagement needed for efficacy, while avoiding both mechanism-based and off-target toxicity. Herein, we report clickable probes based on the XPO1 inhibitors selinexor and eltanexor for the labeling of XPO1 in live cells to assess target engagement and selectivity. We used mass spectrometry-based chemoproteomic workflows to profile the proteome-wide selectivity of selinexor and eltanexor and show that they are highly selective for XPO1. Thermal profiling analysis of selinexor further offers an orthogonal approach to measure XPO1 engagement in live cells. We believe these probes and assays will serve as useful tools to further interrogate the biology of XPO1 and its inhibition in cellular and in vivo systems.

Ribosomal proteins as distinct “passengers” of microvesicles: new semantics in myeloma and mesenchymal stem cells' communication

Aberrant mesenchymal stem cells (MSCs) in multiple myeloma (MM) bone marrows (BM) promote disease progression and drug resistance. Here, we assayed the protein cargo transported from MM-MSCs to MM cells via microvesicles (MVs) with focus on ribosomal proteins (RPs) and assessment of their influence on translation initiation and design of MM phenotype. Proteomics analysis (mass spectrometry) demonstrated increased levels and repertoire of RPs in MM-MSCs MVs compared to normal donors (ND) counterparts (n = 3-8; P = 9.96E - 08). We limited the RPs load in MM-MSCs MVs (starvation, RSK and XPO1 inhibitions), reapplied the modified MVs to MM cell lines (U266, MM1S), and demonstrated that the RPs are essential to the proliferative effect of MM-MSCs MVs on MM cells (n = 3; P < 0.05). We also observed that inhibition with KPT-185 (XPO1 inhibitor) displayed the most extensive effect on RPs delivery into the MVs (↓80%; P = 3.12E - 05). Using flow cytometry we assessed the expression of select RPs (n = 10) in BM-MSCs cell populations (ND and MM; n ≥ 6 each). This demonstrated a heterogeneous expression of RPs in MM-MSCs with distinct subgroups, a phenomenon absent from ND-MSCs samples. These findings bring to light a new mechanism in which the tumor microenvironment participates in cancer promotion. MVs-mediated horizontal transfer of RPs between niche MSCs and myeloma cells is a systemic way to bestow pro-cancer advantages. This capacity also differentiates normal MSCs from the MM-modified MSCs and may mark their reprogramming. Future studies will be aimed at assessing the clinical and therapeutic potential of the increased RPs levels in MM-MSCs MVs.

Sequential Administration of XPO1 and ATR Inhibitors Enhances Therapeutic Response in TP53-mutated Colorectal Cancer

Understanding the mechanisms by which tumors adapt to therapy is critical for developing effective combination therapeutic approaches to improve clinical outcomes for patients with cancer. To identify promising and clinically actionable targets for managing colorectal cancer (CRC), we conducted a patient-centered functional genomics platform that includes approximately 200 genes and paired this with a high-throughput drug screen that includes 262 compounds in four patient-derived xenografts (PDXs) from patients with CRC. Both screening methods identified exportin 1 (XPO1) inhibitors as drivers of DNA damage-induced lethality in CRC. Molecular characterization of the cellular response to XPO1 inhibition uncovered an adaptive mechanism that limited the duration of response in TP53-mutated, but not in TP53-wild-type CRC models. Comprehensive proteomic and transcriptomic characterization revealed that the ATM/ATR-CHK1/2 axes were selectively engaged in TP53-mutant CRC cells upon XPO1 inhibitor treatment and that this response was required for adapting to therapy and escaping cell death. Administration of KPT-8602, an XPO1 inhibitor, followed by AZD-6738, an ATR inhibitor, resulted in dramatic antitumor effects and prolonged survival in TP53-mutant models of CRC. Our findings anticipate tremendous therapeutic benefit and support the further evaluation of XPO1 inhibitors, especially in combination with DNA damage checkpoint inhibitors, to elicit an enduring clinical response in patients with CRC harboring TP53 mutations.