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Raffit Hassan



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    MA12 - New Frontiers from Pathology to Genomics (ID 138)

    • Event: WCLC 2019
    • Type: Mini Oral Session
    • Track: Mesothelioma
    • Presentations: 2
    • Now Available
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      MA12.03 - PARP Inhibitor Sensitivity Does Not Depend on BAP1 but Is Enhanced by Temozolomide in MGMT Deficient Human Mesothelioma Cells (Now Available) (ID 2492)

      14:00 - 15:30  |  Presenting Author(s): Raffit Hassan

      • Abstract
      • Presentation
      • Slides

      Background

      BRCA1 associated protein 1 (BAP1), a nuclear deubiquitinase involved in DNA double-strand (DSB) break repair by homologous recombination (HR), is frequently mutated in mesotheliomas. Because poly (ADP-ribose) polymerase inhibitors (PARPIs) target PARP1 and PARP2 and induce synthetic lethality in BRCA1/2 mutant cancers deficient in HR, we evaluated whether BAP1 inactivating mutations confer sensitivity to PARPIs in mesotheliomas.

      Method

      Ten patient-derived mesothelioma cell lines were generated and characterized for BAP1 mutation status, protein expression and function. Cellular sensitivity to two clinical PARPIs, olaparib and talazoparib were tested as single agents, and in combination with temozolomide. BAP1-deleted mesothelioma cellular models were generated by CRISPR/Cas9 and assessed for sensitivity to PARPIs. Because Schlafen 11 (SLFN11) and O6-methylguanine methyltransferase (MGMT) also drive response to temozolomide and PARPIs, we tested their expression and relationship with drug response.

      Result

      BAP1 inactivating mutations were present in eight of ten cell lines, with two harboring homozygous deletion. Cell lines exhibiting BAP1 expression also showed deubiquitinase activity (DUB). IC50 of olaparib and talazoparib plot classified them into sensitive or resistant population irrespective of BAP1 status (Figure 1). Although BAP1 knockout led to the loss of DUB activity, it did not increase the sensitivity of the cell ines to PARPI. Interestingly, cellular sensitivity to PARPI was increased by temozolomide in MGMT-negative and SLFN11-positive cell lines (Table 1).

      figure 1.jpg

      Figure 1. IC50 of olaparib vs talazoparib – based plot shows a separation of sensitive (red oval) and resistant (blue oval) cell line clusters independent of BAP1 activity.

      table 1.jpg

      Table 1. Summary reflecting combination study between talazoparib and temozolomide in different cell lines having varying MGMT and SLFN11 expression status.

      Conclusion

      BAP1 status does not determine cellular sensitivity to PARPIs in patient-derived mesothelioma cell lines. In MGMT-deficient and SLFN11-positive cells, combination of PARPI and temozolomide is synergistic.

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      MA12.11 - Anti-Tumor Efficacy of Mesothelin Targeted Immunotoxin LMB-100 Plus Pembrolizumab in Mesothelioma Patients and Mouse Models (Now Available) (ID 2316)

      14:00 - 15:30  |  Presenting Author(s): Raffit Hassan

      • Abstract
      • Presentation
      • Slides

      Background

      LMB-100 is an immunotoxin targeting mesothelin (MSLN) that is highly expressed in malignant mesothelioma and lung adenocarcinoma. Given the clinical efficacy of immune checkpoint inhibitors in these cancers, we aimed to evaluate if LMB-100 in combination with αPD-1 antibody will result in greater anti-tumor efficacy.

      Method

      Patients who were treated on a phase I clinical trial of LMB-100 and then received pembrolizumab were evaluated for anti-tumor response and overall survival. We also evaluated LMB-100/αPD-1 combination efficacy in humanized mouse model transplanted with the tumor cells derived from the mesothelioma patient who achieved complete response and healthy donor’s PBMCs. Immune gene expression in pre- and post- LMB-100-treated mesothelioma patient tumor biopsies was detected with NanoString. To further understand the mechanisms of anti-tumor efficacy of LMB-100 plus αPD-1 therapy,we established a human MSLN expressing lung adenocarcinoma syngeneic mouse model with mouse lung adenocarcinoma cell line 531LN2 stably transfected with a vector encoding hMSLN. Using NanoString gene expression assay and flow cytometry, we analyzed drug induced cancer immune responses in 531LN2-hMSLN tumors. Finally, to understand the role of CD8+ T cells in the anti-tumor effects, we depleted CD8+ T cells in LMB-100 plus anti-PD-1 treated 531LN2-hMSLN bearing mice.

      Result

      Nine mesothelioma patients received pembrolizumab, off-protocol, within 3-4 weeks post LMB-100 treatment. Two patients had disease progression before they could be evaluated for tumor response. Out of the 7 patients who were evaluable for response, 4 had durable objective tumor response including 1 complete and 3 partial responses with progression free survival of 104.3+, 49.6, 49.2 and 37.7 weeks. The overall survival of patients with response was 30.2+, 27.7, 23.8+, and 13.8 months from the start of LMB-100 treatments. The immune cell type signature scores including CD45+, CD8+ T cells, exhausted CD8+ T cells, dendritic cells and macrophages were increased in 4 of 6 patients post LMB-100 treatments. The enhanced anti-tumor effects with LMB-100/αPD1 combination were also observed in the PBMC humanized mouse model transplanted with the tumor cells derived the patient with complete response. In the 531LN2-hMSLN mouse syngeneic model, tumor growth was significantly inhibited by LMB-100/αPD-1 treatments than either monotherapy and overall survival was improved in the combination treated mice. The median tumor volume was 865mm3, 420mm3, 277mm3, and 65mm3 in untreated, LMB-100-treated, αPD-1-treated, and combination treated groups respectively on day 34 post tumor inoculation (p<0.001). We observed increased expression of genes related to CD8+T cells and antigen presentation in tumors treated with LMB-100/αPD-1 compared to either agent alone. Flow cytometry confirmed the CD8+T cells increase in LMB-100 /αPD-1 treated 531LN2-hMSLN tumor. Depletion of CD8+T cells significantly negated the anti-tumor benefits in LMB-100/αPD-1-treated mice.

      Conclusion

      Pembrolizumab following LMB-100 is associated with durable tumor response in mesothelioma patients as well as pre-clinical models of mesothelioma and lung cancer. This combination is currently being evaluated in a prospective clinical trial in patients with mesothelioma (clinicaltrials.gov # NCT03644550).

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