Proteasome inhibition targets the KMT2A transcriptional complex in acute lymphoblastic leukemia.

Rearrangments in Histone-lysine-N-methyltransferase 2A (KMT2Ar) are associated with pediatric, adult and therapy-induced acute leukemias. Infants with KMT2Ar acute lymphoblastic leukemia (ALL) have a poor prognosis with an event-free-survival of 38%. Herein we evaluate 1116 FDA approved compounds in primary KMT2Ar infant ALL specimens and identify a sensitivity to proteasome inhibition. Upon exposure to this class of agents, cells demonstrate a depletion of histone H2B monoubiquitination (H2Bub1) and histone H3 lysine 79 dimethylation (H3K79me2) at KMT2A target genes in addition to a downregulation of the KMT2A gene expression signature, providing evidence that it targets the KMT2A transcriptional complex and alters the epigenome. A cohort of relapsed/refractory KMT2Ar patients treated with this approach on a compassionate basis had an overall response rate of 90%. In conclusion, we report on a high throughput drug screen in primary pediatric leukemia specimens whose results translate into clinically meaningful responses. This innovative treatment approach is now being evaluated in a multi-institutional upfront trial for infants with newly diagnosed ALL.

Degradation of Janus kinases in CRLF2-rearranged acute lymphoblastic leukemia.

CRLF2-rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) accounts for more than half of Philadelphia chromosome-like (Ph-like) ALL and is associated with a poor outcome in children and adults. Overexpression of CRLF2 results in activation of Janus kinase (JAK)-STAT and parallel signaling pathways in experimental models, but existing small molecule inhibitors of JAKs show variable and limited efficacy. Here, we evaluated the efficacy of proteolysis-targeting chimeras (PROTACs) directed against JAKs. Solving the structure of type I JAK inhibitors ruxolitinib and baricitinib bound to the JAK2 tyrosine kinase domain enabled the rational design and optimization of a series of cereblon (CRBN)-directed JAK PROTACs utilizing derivatives of JAK inhibitors, linkers, and CRBN-specific molecular glues. The resulting JAK PROTACs were evaluated for target degradation, and activity was tested in a panel of leukemia/lymphoma cell lines and xenograft models of kinase-driven ALL. Multiple PROTACs were developed that degraded JAKs and potently killed CRLF2r cell lines, the most active of which also degraded the known CRBN neosubstrate GSPT1 and suppressed proliferation of CRLF2r ALL in vivo, e.g. compound 7 (SJ988497). Although dual JAK/GSPT1-degrading PROTACs were the most potent, the development and evaluation of multiple PROTACs in an extended panel of xenografts identified a potent JAK2-degrading, GSPT1-sparing PROTAC that demonstrated efficacy in the majority of kinase-driven xenografts that were otherwise unresponsive to type I JAK inhibitors, e.g. compound 8 (SJ1008030). Together, these data show the potential of JAK-directed protein degradation as a therapeutic approach in JAK-STAT-driven ALL and highlight the interplay of JAK and GSPT1 degradation activity in this context.

Modeling and targeting of erythroleukemia by hematopoietic genome editing.

Acute erythroid leukemia (AEL) is characterized by a distinct morphology, mutational spectrum, lack of preclinical models, and poor prognosis. Here, using multiplexed genome editing of mouse hematopoietic stem and progenitor cells and transplant assays, we developed preclinical models of AEL and non-erythroid acute leukemia and describe the central role of mutational cooperativity in determining leukemia lineage. Different combination of mutations in Trp53, Bcor, Dnmt3a, Rb1, and Nfix resulted in the development of leukemia with an erythroid phenotype, accompanied by the acquisition of alterations in signaling and transcription factor genes that recapitulate human AEL by cross-species genomic analysis. Clonal expansion during tumor evolution was driven by mutational cooccurrence, with clones harboring a higher number of founder and secondary lesions (eg, mutations in signaling genes) showing greater evolutionary fitness. Mouse and human AEL exhibited deregulation of genes regulating erythroid development, notably Gata1, Klf1, and Nfe2, driven by the interaction of mutations of the epigenetic modifiers Dnmt3a and Tet2 that perturbed methylation and thus expression of lineage-specific transcription factors. The established mouse leukemias were used as a platform for drug screening. Drug sensitivity was associated with the leukemia genotype, with the poly (ADP-ribose) polymerase inhibitor talazoparib and the demethylating agent decitabine efficacious in Trp53/Bcor-mutant AEL, CDK7/9 inhibitors in Trp53/Bcor/Dnmt3a-mutant AEL, and gemcitabine and bromodomain inhibitors in NUP98-KDM5A leukemia. In conclusion, combinatorial genome editing has shown the interplay of founding and secondary genetic alterations in phenotype and clonal evolution, epigenetic regulation of lineage-specific transcription factors, and therapeutic tractability in erythroid leukemogenesis.

Bidirectional immune tolerance in nonmyeloablative MHC-mismatched BMT for murine ß-thalassemia.

Nonmyeloablative conditioning using total lymphoid irradiation (TLI) and rabbit antithymocyte serum (ATS) (the murine preclinical equivalent of antithymocyte globulin [ATG]) facilitates immune tolerance after bone marrow transplantation (BMT) across major histocompatibility complex (MHC) disparities and may be a useful strategy for nonmalignant disorders. We previously reported that donor effector T-cell function and graft-versus-host disease (GVHD) are regulated via recipient invariant natural killer T-cell (iNKT) interleukin-4-driven expansion of donor Foxp3+ naturally occurring regulatory T cells (Tregs). This occurs via recipient iNKT- and STAT6-dependent expansion of recipient myeloid dendritic cells (MDCs) that induce contact-dependent expansion of donor Treg through PD-1/PD ligand signaling. After TLI/ATS + BMT, Gr-1lowCD11c+ MDCs and Gr-1highCD11cneg myeloid-derived suppressor cells (MDSCs) were enriched in GVHD target organs. We now report that the recovery of both recipient MDSCs (P < .01) and MDCs (P < .01) is significantly increased when the alkylator cyclophosphamide (CTX) is added to TLI/ATS conditioning. In a BALB/c → B6 lethal GVHD model, adoptive transfer of MDSCs from TLI/ATS/CTX-conditioned recipients is associated with significantly improved GVHD colitis and survival (P < .001), conversion of MDSCs to PD ligand-expressing MDCs, and increased donor naturally occurring Treg recovery (P < .01) compared with control treatment. Using BALB/c donors and ß-thalassemic HW-80 recipients, we found significantly improved rates of engraftment and GVHD following TLI/ATS/CTX compared with TLI/ATS, lethal or sublethal total body irradiation/ATS/CTX, or CTX/ATS conditioning. These data provide preclinical support for trials of TLI/ATG/alkylator regimens for MHC-mismatched BMT for hemoglobinopathies. The data also delineate innate immune mechanisms by which TLI/ATS/CTX conditioning may augment transplantation tolerance.

Recipient myeloid-derived immunomodulatory cells induce PD-1 ligand-dependent donor CD4+Foxp3+ regulatory T cell proliferation and donor-recipient immune tolerance after murine nonmyeloablative bone marrow transplantation.

We showed previously that nonmyeloablative total lymphoid irradiation/rabbit anti-thymocyte serum (TLI/ATS) conditioning facilitates potent donor-recipient immune tolerance following bone marrow transplantation (BMT) across MHC barriers via recipient invariant NKT (iNKT) cell-derived IL-4-dependent expansion of donor Foxp3(+) naturally occurring regulatory T cells (nTregs). In this study, we report a more specific mechanism. Wild-type (WT) BALB/c (H-2(d)) hosts were administered TLI/ATS and BMT from WT or STAT6(-/-) C57BL/6 (H-2(b)) donors. Following STAT6(-/-) BMT, donor nTregs demonstrated no loss of proliferation in vivo, indicating that an IL-4-responsive population in the recipient, rather than the donor, drives donor nTreg proliferation. In graft-versus-host disease (GVHD) target organs, three recipient CD11b(+) cell subsets (Gr-1(high)CD11c(-), Gr-1(int)CD11c(-), and Gr-1(low)CD11c(+)) were enriched early after TLI/ATS + BMT versus total body irradiation/ATS + BMT. Gr-1(low)CD11c(+) cells induced potent H-2K(b+)CD4(+)Foxp3(+) nTreg proliferation in vitro in 72-h MLRs. Gr-1(low)CD11c(+) cells were reduced significantly in STAT6(-/-) and iNKT cell-deficient Jα18(-/-) BALB/c recipients after TLI/ATS + BMT. Depletion of CD11b(+) cells resulted in severe acute GVHD, and adoptive transfer of WT Gr-1(low)CD11c(+) cells to Jα18(-/-) BALB/c recipients of TLI/ATS + BMT restored day-6 donor Foxp3(+) nTreg proliferation and protection from CD8 effector T cell-mediated GVHD. Blockade of programmed death ligand 1 and 2, but not CD40, TGF-ß signaling, arginase 1, or iNOS, inhibited nTreg proliferation in cocultures of recipient-derived Gr-1(low)CD11c(+) cells with donor nTregs. Through iNKT-dependent Th2 polarization, myeloid-derived immunomodulatory dendritic cells are expanded after nonmyeloablative TLI/ATS conditioning and allogeneic BMT, induce PD-1 ligand-dependent donor nTreg proliferation, and maintain potent graft-versus-host immune tolerance.

Topoisomerase assays.

Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I enzymes, which make single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are for assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA, and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay to examine topoisomerase covalent complexes in vivo, and an assay for measuring DNA cleavage in vitro.

Aging affects response to cyclic tensile stretch: paradigm for intervertebral disc degeneration.

Much evidence supports a fundamental role for mechanical forces in modulating differentiation, homeostasis, and remodelling of musculoskeletal cells. Little is known, however, regarding mechanobiology and gene expression of intervertebral disc (IVD) cells from older individuals. To characterise the effect of mechanical stimulation on cells from older discs, an in vitro study of IVD cells harvested from different aged pigs was conducted to measure extracellular matrix (ECM) gene expression in response to cyclic tensile stress (CTS). Gene expression of annulus fibrosus (AF) cells from IVDs of mature and older pigs was quantified for the predominant ECM genes; type I collagen, type II collagen and aggrecan, and matrix metalloproteinase 1 (MMP-1), a collagenase that degrades fibrillar collagens. AF cells cultured on flexible-bottom plates were stretched 10 % at 0.5 Hz frequency. After 24 h, gene expression was assayed using reverse transcriptase polymerase chain reaction (RT-PCR). Basal mRNA levels without stretching for type II collagen and aggrecan were lower in older annular cells whereas MMP-1 levels were higher compared to mature cells. Following CTS, an adaptive response was elicited in annular cells from both age groups. ECM protein genes were upregulated, whereas MMP-1 was downregulated. The magnitude of response was significantly greater in older cells as compared to mature cells. These data suggest that the cells from the AF of older animals manifest lower basal levels of mRNA for type II collagen and aggrecan and higher levels of MMP-1 possibly due to decreased tensile stress experienced in vivo and is not the result of reduced capacity for response.

End-processing during non-homologous end-joining: a role for exonuclease 1.

Non-homologous end-joining (NHEJ) is a critical error-prone pathway of double strand break repair. We recently showed that tyrosyl DNA phosphodiesterase 1 (Tdp1) regulates the accuracy of NHEJ repair junction formation in yeast. We assessed the role of other enzymes in the accuracy of junction formation using a plasmid repair assay. We found that exonuclease 1 (Exo1) is important in assuring accurate junction formation during NHEJ. Like tdp1Δ mutants, exo1Δ yeast cells repairing plasmids with 5'-extensions can produce repair junctions with templated insertions. We also found that exo1Δ mutants have a reduced median size of deletions when joining DNA with blunt ends. Surprisingly, exo1Δ pol4Δ mutants repair blunt ends with a very low frequency of deletions. This result suggests that there are multiple pathways that process blunt ends prior to end-joining. We propose that Exo1 acts at a late stage in end-processing during NHEJ. Exo1 can reverse nucleotide additions occurring due to polymerization, and may also be important for processing ends to expose microhomologies needed for NHEJ. We propose that accurate joining is controlled at two steps, a first step that blocks modification of DNA ends, which requires Tdp1, and a second step that occurs after synapsis that requires Exo1.