Papers by Makhdum Ahmed, MD, PhD
Blood, Nov 29, 2018
Introduction The majority of secondary central nervous system (CNS) lymphoma cases are derived fr... more Introduction The majority of secondary central nervous system (CNS) lymphoma cases are derived from histologically high-grade systemic lymphomas such as diffuse-large B cell lymphoma (DLBCL) and Burkitt's lymphoma. These CNS lymphomas are associated with poor prognosis and a low likelihood of long-term survival, especially rituximab-ineffective CD20- lymphoma. To investigate the biological features and potential therapeutic targets of CNS-involved CD20- lymphoma, we established the first known patient-derived xenograft (PDX) model using cerebrospinal fluid (CSF). Methods Six- to 8-week-old male NSG mice (Jackson Laboratory) were housed and monitored in our animal research facility. All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center. A total of 0.08 million cells were isolated from 2 mL of CSF obtained from a CD20- CD79A- DLBCL patient with Bcl-2 and Myc rearrangements (double-hit). The isolated cells were directly injected into a human fetal bone chip implanted into NSG (NSG-hu) mice. After passage, the mice were administered several small molecules, and tumor burden was measured via tumor volume and human β2M levels over the course of treatment. Kaplan-Meier survival analysis was also performed. PDX tissue was collected for histological analysis, immunophenotyping, genotyping, and proteomics analysis. Results The PDX tumors possessed the same histological, genetic and immunophenotypical features as the primary patient tumor cells. The PDXs had Bcl-2 and Myc rearrangements and were positive for CD10, CD19, CD22, and CD45 and were negative for CD5, CD20, and CD79A. After tumor passage, the mice were treated with vehicle, 50 mg/kg ibrutinib (Bruton's tyrosine kinase (BTK) inhibitor) oral gavage daily, 50 mg/kg ABT199 (Bcl-2 inhibitor) oral gavage daily, 1 mg/kg carfilzomib (proteasome inhibitor) i.v. twice per week, or 7.5 mg/kg IACS-010759 (oxidative phosphorylation (OXPHOS) inhibitor) oral gavage daily. As shown in Figure 1, the PDX tumor was resistant to the BTK inhibitor ibrutinib and moderately responsive to the proteasome inhibitor carfilzomib. Although the Bcl-2 gene was rearranged, the PDX model only had a partial response to the Bcl-2 inhibitor ABT-199 (ABT-199 vs control, p = 0.0004). In contrast, inhibition of the OXPHOS pathway with IACS-010759 significantly inhibited 96% tumor growth (IACS-010759 vs control, p < 0.00001) and prolonged mouse survival by approximately 25 days compared with the vehicle control and the other drugs (IACS-010759 vs control, ABT-199, or ibrutinib, p < 0.0001). Moreover, proteomics analysis demonstrated that IACS-010759 not only inhibited OXPHOS but also upregulated the pro-apoptotic protein Bim. Conclusion To our knowledge, this is the first PDX model generated from the CSF of a CD20- double-hit DLBCL patient. With this PDX model, we determined that the OXPHOS inhibitor IACS-010759 is a promising therapeutic agent for this aggressive cancer. Disclosures Wang: Acerta Pharma: Honoraria, Research Funding; Kite Pharma: Research Funding; Novartis: Research Funding; Pharmacyclics: Honoraria, Research Funding; Dava Oncology: Honoraria; Juno: Research Funding; MoreHealth: Consultancy; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
Cancer Research, Jul 1, 2017
Mantle cell lymphoma (MCL) accounts for 6-8% of all non-Hodgkin lymphoma cases and is a therapeut... more Mantle cell lymphoma (MCL) accounts for 6-8% of all non-Hodgkin lymphoma cases and is a therapeutic challenge. MCL is characterized by the expression of different B-cell markers such as CD-19, CD-20 and BSAP/PAX5, and CD-20 is strongly expressed and can be used as a potential target. MT-3724 was developed by Molecular Templates and is an engineered toxin body (ETB) targeting CD-20. MT-3724 binds CD-20 and forces its own internalization into the target cell where it subsequently self-routes to the cytosol to enzymatically and permanently inhibit protein synthesis via ribosome inactivation. By selectively and specifically targeting CD20-positive cells, MT-3724 may decrease cell proliferation and induce apoptosis in MCL. We tested the effects of MT-3724 by in vitro cell proliferation in 3 ibrutinib-sensitive cell lines and 5 ibrutinib-resistant cell lines (4 primary resistant and 1 acquired resistant). We also measured the levels of apoptotic cells in both ibrutinib-sensitive and -resistant cell lines treated with MT-3724 by Annexin V/ PI staining. Lastly, we conducted an in vivo efficacy assay of MT-3724 in a MCL PDX model resistant to a wide-range of drugs, including ibrutinib. MT-3724 inhibited cell proliferation effectively and efficiently in most ibrutinib-sensitive and ibrutinib-resistant cell lines in a dose-dependent manner. IC50 < 500 ng/ml was characterized as sensitive to MT-3724, and IC50 > 500 ng/ml was considered resistant to MT-3724. Regarding the ibrutinib-sensitive cell lines, the 3 cell lines (Jeko-1, Mino and Rec-1) were sensitive to MT-3724 with IC50 values of 139.1, 309.3 and 457.7 ng/ml, respectively. Regarding the ibrutinib-resistant cell lines, 4 cell lines (Maver-1, JVM-13, Jeko-R and Granta519) were sensitive to MT-3724 with IC50 values of 124.6, 155.1, 266.2 and 442.4 ng/ml, respectively, and 1 cell line (Z-138) was resistant to MT-3724 (IC50 = 1231 ng/ml). However, no significant differences in IC50 values were found between ibrutinib-sensitive and -resistant cell lines (p = 0.36). In a time-dependent assay, 300 ng/ml MT-3724 also reduced cell proliferation in 2 ibrutinib-sensitive cell lines (Mino and Jeko-1) and 2 ibrutinib-resistant cell lines (Jeko-R and Maver-1) over time. Furthermore, MT-3724 also induced cell apoptosis in both ibrutinib-sensitive (Jeko-1) and -resistant (Jeko-R and Maver-1) cell lines. Lastly, MT-3724 was administered intraperitoneally for three consecutive weeks in a PDX model resistant to a wide-range of targeted agents. Interestingly, MT-3724 dramatically reduced tumor burden and increased survival (median of 27 days) of the PDX mice. MT-3724 is the first toxin engineered body targeting CD-20 used in MCL, which may be a potential therapeutic candidate for MCL, especially for drug-resistant cases. Citation Format: Shengjian Huang, Taylor Bell, Yang Liu, Hui Guo, Carrie Li, Makhdum Ahmed, Laura Lam, Hui Zhang, Zhihong Chen, Michael L. Wang, Leo Zhang, Krystle Nomie. Preclinical examination of the effects of MT-3724, an engineered toxin body targeting CD20, in mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3651. doi:10.1158/1538-7445.AM2017-3651
A, Schematic describing the primary B-cell lymphoma PDX SCID/NSG-hu mouse model. Six-to 8-week-ol... more A, Schematic describing the primary B-cell lymphoma PDX SCID/NSG-hu mouse model. Six-to 8-week-old male NSG or CB-17 SCID mice were implanted with human fetal bone. Approximately 4 to 6 weeks following bone implantation, one injection of 5 Ã- 106 patient tumor cells were administered directly into the human fetal bone chip as the first generation (G1). The levels of circulating human β2M in mouse serum were used to monitor tumor engraftment and growth in G1 mice. Once tumor burden was detected, the pieces of tumor with bone chip were subcutaneously transplanted into NSG mice as the second generation (G2). After G2, the consistent growths of tumor in NSG mice were acquired. Single-agent targeted therapy, drug-resistant treatment, or combination therapy, were administered in the indicated generations. B, Human β2M reflected the tumor burdens of 16 B-cell lymphomas in PDX-G1 mice. After 3-4 weeks of tumor inoculation, mouse sera were collected from the peripheral blood of tail vein, and the level of human β2M was determined by ELISA.
Freshly isolated lymphoma cells from PT2-BL-G6 and G7 PDXs. Cell viability was tested by CellTite... more Freshly isolated lymphoma cells from PT2-BL-G6 and G7 PDXs. Cell viability was tested by CellTiter-Glo luminescent cell viability assay after 48-hour incubation with the indicated drug treatment. The in vitro drug and dose information for IBN and ABT-199 are listed in Supplementary Table S1. IBN, ibrutinib; PT, patient; BL, Burkitt's lymphoma.
Supplementary Table S3. OncoPlus Gene List and Identified Variants
PT8 was clinically primary ibrutinib resistant. PT8-MCL PDX was generated and passaged. G3 mice w... more PT8 was clinically primary ibrutinib resistant. PT8-MCL PDX was generated and passaged. G3 mice were treated with vehicle control, IBN, and CFZ (P < 0.01, CFZ versus vehicle control or IBN, n = 5).
Include supplementary Figure S1-S4 legends; Supplementary Table S1 and S4; and list Supplementary... more Include supplementary Figure S1-S4 legends; Supplementary Table S1 and S4; and list Supplementary Table S2 and S3 titles. Supplementary Table S1. The panel of drugs used in vitro and in vivo with their doses and duration. Supplementary Table S4. The mean passage time per generation for 5 generations
PT8 was clinically primary ibrutinib resistant. PT8-MCL PDX was generated and passaged. G3 mice w... more PT8 was clinically primary ibrutinib resistant. PT8-MCL PDX was generated and passaged. G3 mice were treated with vehicle control, IBN, and CFZ (P < 0.01, CFZ versus vehicle control or IBN, n = 5).
A, Schematic describing the primary B-cell lymphoma PDX SCID/NSG-hu mouse model. Six-to 8-week-ol... more A, Schematic describing the primary B-cell lymphoma PDX SCID/NSG-hu mouse model. Six-to 8-week-old male NSG or CB-17 SCID mice were implanted with human fetal bone. Approximately 4 to 6 weeks following bone implantation, one injection of 5 Ã- 106 patient tumor cells were administered directly into the human fetal bone chip as the first generation (G1). The levels of circulating human β2M in mouse serum were used to monitor tumor engraftment and growth in G1 mice. Once tumor burden was detected, the pieces of tumor with bone chip were subcutaneously transplanted into NSG mice as the second generation (G2). After G2, the consistent growths of tumor in NSG mice were acquired. Single-agent targeted therapy, drug-resistant treatment, or combination therapy, were administered in the indicated generations. B, Human β2M reflected the tumor burdens of 16 B-cell lymphomas in PDX-G1 mice. After 3-4 weeks of tumor inoculation, mouse sera were collected from the peripheral blood of tail vein, ...
Supplementary Table S3. OncoPlus Gene List and Identified Variants
Freshly isolated lymphoma cells from PT2-BL-G6 and G7 PDXs. Cell viability was tested by CellTite... more Freshly isolated lymphoma cells from PT2-BL-G6 and G7 PDXs. Cell viability was tested by CellTiter-Glo luminescent cell viability assay after 48-hour incubation with the indicated drug treatment. The in vitro drug and dose information for IBN and ABT-199 are listed in Supplementary Table S1. IBN, ibrutinib; PT, patient; BL, Burkitt's lymphoma.
Include supplementary Figure S1-S4 legends; Supplementary Table S1 and S4; and list Supplementary... more Include supplementary Figure S1-S4 legends; Supplementary Table S1 and S4; and list Supplementary Table S2 and S3 titles. Supplementary Table S1. The panel of drugs used in vitro and in vivo with their doses and duration. Supplementary Table S4. The mean passage time per generation for 5 generations
Representative tumor mass and histology of tumor series passages of PT1 MCL and PT2 Burkitt's... more Representative tumor mass and histology of tumor series passages of PT1 MCL and PT2 Burkitt's lymphoma (BL). B-C, PDX tumors invaded the mouse spleen and formed splenomegaly. D, Patient abdomen PET/CT images of PT1 and PT5 showed splenomegaly (left panel). Autopsy of PT1 G3 NSG mouse and PT5 G1 SCID-hu mouse also showed splenomegaly (middle panel), and H&E staining of mouse spleens in PDXs indicated extensive lymphoma involvement (right panel).
Purpose: Patients with B-cell lymphomas often relapse after frontline therapy, and novel therapie... more Purpose: Patients with B-cell lymphomas often relapse after frontline therapy, and novel therapies are urgently needed to provide long-term remission. We established B-cell lymphoma patient-derived xenograft (PDX) models to assess their ability to mimic tumor biology and to identify B-cell lymphoma patient treatment options.Experimental Design: We established the PDX models from 16 patients with diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, or Burkitt lymphoma by inoculating the patient tumor cells into a human bone chip implanted into mice. We subjected the PDX models to histopathologic and phenotypical examination, sequencing, and drug efficacy analysis. Primary and acquired resistance to ibrutinib, an oral covalent inhibitor of Bruton tyrosine kinase, were investigated to elucidate the mechanisms underlying ibrutinib resistance and to identify drug treatments to overcome resistance.Results: The PDXs maintained the same biologica...
Blood, 2016
Background: Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy that is initially respo... more Background: Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy that is initially responsive but ultimately relapses to frontline therapy. Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of…
Influenza and other respiratory viruses, Jan 2, 2018
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Papers by Makhdum Ahmed, MD, PhD