Research ArticleCancer

Overcoming mutational complexity in acute myeloid leukemia by inhibition of critical pathways

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Science Translational Medicine  25 Oct 2017:
Vol. 9, Issue 413, eaao1214
DOI: 10.1126/scitranslmed.aao1214
  • Fig. 1. In vivo fates of patient-derived AML cells defined by mutational profile.

    (A) Somatic mutation profiles were identified in patient cell subpopulations defined by surface phenotype based on developmental hierarchy of human hematopoiesis. (B) The in vivo fate of each subpopulation was determined through transplantation into newborn NSG mice. If repopulation by multilineage hematopoiesis occurred, then the transplanted subpopulation contained hematopoietic or preleukemic stem cells; if AML engraftment occurred, then the transplanted subpopulation contained LICs.

  • Fig. 2. Mutational profiles and in vivo engraftment of patient-derived subpopulations.

    Three additional patients are shown in fig. S1. In each patient sample, human CD45+CD3+CD19 T cells and human CD45+CD3CD19+ B cells were identified. Within CD3CD19 non-T, non-B cells, subpopulations were identified on the basis of CD34, CD38, CD90, and CD45RA surface expression. These populations underwent polymerase chain reaction (PCR) for FLT3-ITD mutation and DNA sequencing (DNA-seq) for the other genes indicated. Variant allele frequencies are shown as heat maps. In patients 21 (A) and 20 (B), the CD34+CD38CD90CD45RA and CD34+CD38CD90CD45RA+ subpopulations were identified. The in vivo fates of CD34+CD38CD90CD45RA subpopulations differed between patients 20 and 21, showing engraftment with multilineage human hematopoiesis in patient 20 (indicated by green rectangles) but initiation of AML in patient 21 (indicated by red rectangles). In patient 13 (C), the CD34+CD38 subpopulation showed multilineage repopulation, whereas the CD34CD33+ subpopulation with additional FLT3-ITD and NPM1 mutations initiated AML. AML-engrafted recipients showed no B cell engraftment (indicated by gray dashed outlines on flow cytometry plots). Detailed information on variants found in each patient is shown in table S2.

  • Fig. 3. Mutations contributing to distinct in vivo cell fates identified by single-cell functional genomics.

    (A) Patient-derived subpopulations with defined in vivo fates and their in vivo progeny underwent single-cell mutation profiling. Through this strategy, mutations present in patient-derived preleukemic stem cells (pre-LSCs) and LIC clones were tracked and linked to in vivo fates. (B and C) Using samples from five patients, patient-derived multilineage-engrafting and LIC-containing population and engrafted B cells and AML cells were subjected to single-cell DNA sequencing for variants detected in each indicated gene in each patient. FLT3-ITD sequences with highly variable repeated sequence patterns were detected by single-cell PCR. In (B) and (C), each column of rectangles represents an individual cell. The presence or absence of mutations in each gene is shown by colors of rectangles, as indicated.

  • Fig. 4. Profiles of AML-associated mutations in nine genes in patient- and recipient-derived AML cells from 12 AML cases.

    Variant allele frequencies of indicated genes are represented as heat maps. Patient, LIC-containing population from the patient; 1′, human AML cells from primary recipients; 2′, human AML cells from secondary recipients. All patient-derived and recipient-derived leukemia populations were positive for FLT3-ITD by PCR. Non-ITD FLT3 mutations were identified by sequencing. Information on variants is shown in table S2.

  • Fig. 5. Induction of apoptosis via enhanced BCL-2 dependence in FLT3-ITD+ AML cells with diverse coexisting somatic mutations by in vivo kinase inhibition.

    Overall, treatment with RK-20449 resulted in significant reduction of AML cells in the BM, spleen, and PB of recipients (P < 1 × 10−19 for each; data are tabulated in table S3). To document patient-to-patient variability, RK-20449 responses were classified as follows: complete response (A), if all recipients treated showed residual BM human CD45+ chimerism of <5%; good response (B), if the case did not meet the criterion for complete responder but all recipients showed <50% residual BM human CD45+; partial response (C), if at least one of the recipients showed >50% residual BM human CD45+. For each response group, PB time course of human AML chimerism (leftmost panels) for RK-20449–treated recipients and final BM (middle panels) and spleen (rightmost panels) human AML chimerism of RK-20449–treated and untreated recipients are shown. Pretreatment PB human AML cell chimerism is shown at week 0. The numbers of recipients for each patient/each treatment group and pre-/posttreatment AML chimerism are shown in table S3. In all response groups, BM, spleen, and PB chimerism was significantly reduced with RK-20449 treatment. For the BM and spleen, final chimerism for recipients in each treatment group was compared. For the PB, pre- and posttreatment chimerism for RK-20449–treated recipients was compared. P < 5.8 × 10−5 by two-tailed t test for all comparisons. In each scatter graph, dotted lines are drawn at 0, 5, and 50%. (D) Dynamics of apoptotic response to BIM peptide in the presence of RK-20449 was measured for six AML cases. Bars represent the BIM IC50 of cytochrome c loss in RK-20449–treated human CD45+ cells as percentages of IC50 in cells treated with dimethyl sulfoxide (DMSO) alone. Enhancement of apoptotic response to BIM by RK-20449 showed substantial patient-to-patient variability. (E) Dynamics of apoptotic response to BH3-only peptides BAD, HRK, and NOXA in the presence of RK-20449 or DMSO alone was measured for seven AML cases. Bars represent the percentage reduction of IC50 in the presence of RK-20449 compared with DMSO alone. RK-20449 enhanced apoptotic response to BAD and HRK peptides with substantial patient-to-patient variability, whereas apoptotic response to NOXA peptide was not substantially enhanced by RK-20449. (F) Apoptotic response to a BCL-2 inhibitor ABT-199 was enhanced by RK-20449 in seven AML cases. Bars show increased cytochrome c loss in human AML cells treated with ABT-199 and RK-20449 compared with those treated with DMSO alone.

  • Fig. 6. Eradication of FLT3-ITD+ AML cells in vivo through combined inhibition of kinase and antiapoptotic pathways.

    (A and B) Mice engrafted with AML derived from 12 patients associated with good or partial responses to RK-20449 alone received four different treatments (no treatment, ABT-199 alone, RK-20449 alone, and combination). The numbers of recipients for each patient/each treatment group and pre-/posttreatment AML chimerism are shown in table S4. (A) PB human CD45+ chimerism is shown over time. Recipients were phlebotomized weekly, and pretreatment PB human CD45+ AML chimerism is shown at time 0. Mean PB human CD45+ chimerism for each patient/each treatment group and statistics comparing the treatment groups are shown in table S5. (B) Final BM and spleen human AML chimerism is shown for mice engrafted with AML derived from 12 patients. Nine cases showed complete responses, and three cases showed good responses to combination treatment. Each circle represents an AML-engrafted recipient. Mean BM/spleen human CD45+ chimerism for each patient/each treatment group and statistics comparing the treatment groups are shown in table S6. In each scatter graph, dotted lines are drawn at 0, 5, and 50%. (C) Residual human AML initiation capacity of human CD45+ cells after in vivo treatment was assessed by serial transplantation for four treatment groups. To compare the amount of residual LICs in recipients after in vivo treatment, each secondary recipient was transplanted with human CD45+ cells sorted from 2.5% (by cell number) of viable BM cells remaining in treated recipients. The mean and SEM for human CD45+ AML cell chimerism in the BM of secondary recipients are shown. Each circle represents a secondary recipient.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/413/eaao1214/DC1

    Fig. S1. Identification of hematopoietic subpopulations in patient samples by surface phenotype and in vivo function in three additional patients.

    Fig. S2. Mutational profiles of AML cells with and without in vivo RK-20449 treatment.

    Fig. S3. Mutational profiles of AML patient samples obtained serially during clinical course.

    Fig. S4. Altered transcription profiles of human AML cells with in vivo RK-20449 treatment.

    Fig. S5. Overcoming genetically complex AML by targeting leukemia-initiating mutation and antiapoptotic BCL-2 pathway.

    Table S1. Patient characteristics (provided as an Excel file).

    Table S2. Information of identified variants (provided as an Excel file).

    Table S3. Human AML chimerism in the PB, BM, and spleen of AML-engrafted recipients treated with RK-20449 alone (provided as an Excel file).

    Table S4. Human AML chimerism in the PB, BM, and spleen of AML-engrafted recipients treated with ABT-199 alone, RK-20449 alone, or combination (provided as an Excel file).

    Table S5. Statistics comparing pre- and posttreatment PB human AML chimerism in recipients treated with ABT-199 alone, RK-20449 alone, or combination (provided as an Excel file).

    Table S6. Statistics comparing BM and spleen human AML chimerism in recipients treated with ABT-199 alone, RK-20449 alone, or combination (provided as an Excel file).

    Table S7. Effect of in vivo exposure to combined RK-20449 and ABT-199 on human multilineage hematopoiesis (provided as an Excel file).

    Table S8. PCR primers for targeted sequencing of MALBAC products (provided as an Excel file).

    Reference (45)

  • Supplementary Material for:

    Overcoming mutational complexity in acute myeloid leukemia by inhibition of critical pathways

    Yoriko Saito, Yoshiki Mochizuki, Ikuko Ogahara, Takashi Watanabe, Leah Hogdal, Shinsuke Takagi, Kaori Sato, Akiko Kaneko, Hiroshi Kajita, Naoyuki Uchida, Takehiro Fukami, Leonard D. Shultz, Shuichi Taniguchi, Osamu Ohara, Anthony G. Letai, Fumihiko Ishikawa*

    *Corresponding author. Email: fumihiko.ishikawa{at}riken.jp

    Published 25 October 2017, Sci. Transl. Med. 9, eaao1214 (2017)
    DOI: 10.1126/scitranslmed.aao1214

    This PDF file includes:

    • Fig. S1. Identification of hematopoietic subpopulations in patient samples by surface phenotype and in vivo function in three additional patients.
    • Fig. S2. Mutational profiles of AML cells with and without in vivo RK-20449 treatment.
    • Fig. S3. Mutational profiles of AML patient samples obtained serially during clinical course.
    • Fig. S4. Altered transcription profiles of human AML cells with in vivo RK-20449 treatment.
    • Fig. S5. Overcoming genetically complex AML by targeting leukemia-initiating mutation and antiapoptotic BCL-2 pathway.
    • Legends for tables S1 to S8
    • Reference (45)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Patient characteristics (provided as an Excel file).
    • Table S2. Information of identified variants (provided as an Excel file).
    • Table S3. Human AML chimerism in the PB, BM, and spleen of AML-engrafted recipients treated with RK-20449 alone (provided as an Excel file).
    • Table S4. Human AML chimerism in the PB, BM, and spleen of AML-engrafted recipients treated with ABT-199 alone, RK-20449 alone, or combination (provided as an Excel file).
    • Table S5. Statistics comparing pre- and posttreatment PB human AML chimerism in recipients treated with ABT-199 alone, RK-20449 alone, or combination (provided as an Excel file).
    • Table S6. Statistics comparing BM and spleen human AML chimerism in recipients treated with ABT-199 alone, RK-20449 alone, or combination (provided as an Excel file).
    • Table S7. Effect of in vivo exposure to combined RK-20449 and ABT-199 on human multilineage hematopoiesis (provided as an Excel file).
    • Table S8. PCR primers for targeted sequencing of MALBAC products (provided as an Excel file).

    [Download Tables S1 to S8]

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