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Dysregulation of the BCL-2 family is implicated in protecting chronic myeloid leukemia (CML) cells from intracellular damage and BCR::ABL1-inhibition with tyrosine kinase inhibitors (TKIs) and may be a viable therapeutic target in blast phase (BP-)CML, for which treatment options are limited. BH3 mimetics, a class of small molecule inhibitors with high-specificity against the prosurvival members of the BCL-2 family, have displayed clinical promise in the treatment of chronic lymphocytic and acute myeloid leukemia as single agents and in combination with standard-of-care therapies. Here we present the first comparison of inhibition of BCL-2 prosurvival proteins BCL-2, BCL-xL and MCL-1 in combination with a second or third generation TKI, crucially with comparisons drawn between myeloid and lymphoid BP-CML samples. Co-treatment of four BP-CML cell lines with the TKIs nilotinib or ponatinib and either BCL-2 (venetoclax), MCL-1 (S63845) or BCL-xL (A-1331852) inhibitors resulted in a synergistic reduction in cell viability and increase in phosphatidylserine (PS) presentation. Nilotinib with BH3 mimetic combinations in myeloid BP-CML patient samples triggered increased induction of apoptosis over nilotinib alone, and a reduction in colony-forming capacity and CD34 + fraction, while this was not the case for lymphoid BP-CML samples tested. While some heterogeneity in apoptotic response was observed between cell lines and BP-CML patient samples, the combination of BCL-xL and BCR::ABL1 inhibition was consistently effective in inducing substantial apoptosis. Further, while BH3 mimetics showed little efficacy as single agents, dual-inhibition of BCL-2 prosurvival proteins dramatically induced apoptosis in all cell lines tested and in myeloid BP-CML patient samples compared to healthy donor samples. Gene expression and protein level analysis suggests a protective upregulation of alternative BCL-2 prosurvival proteins in response to BH3 mimetic single-treatment in BP-CML. Our results suggest that BH3 mimetics represent an interesting avenue for further exploration in myeloid BP-CML, for which alternative treatment options are desperately sought., (© 2022. The Author(s).)

Over the last 50 years, there has been a steady improvement in the treatment outcome of acute myeloid leukemia (AML). However, median survival in the elderly is still poor due to intolerance to intensive chemotherapy and higher numbers of patients with adverse cytogenetics. Fadraciclib (CYC065), a novel cyclin-dependent kinase (CDK) 2/9 inhibitor, has preclinical efficacy in AML. In AML cell lines, myeloid cell leukemia 1 (MCL-1) was downregulated following treatment with fadraciclib, resulting in a rapid induction of apoptosis. In addition, RNA polymerase II (RNAPII)-driven transcription was suppressed, rendering a global gene suppression. Rapid induction of apoptosis was observed in primary AML cells after treatment with fadraciclib for 6-8 h. Twenty-four hours continuous treatment further increased efficacy of fadraciclib. Although preliminary results showed that AML cell lines harboring KMT2A rearrangement (KMT2A-r) are more sensitive to fadraciclib, we found that the drug can induce apoptosis and decrease MCL-1 expression in primary AML cells, regardless of KMT2A status. Importantly, the diversity of genetic mutations observed in primary AML patient samples was associated with variable response to fadraciclib, confirming the need for patient stratification to enable a more effective and personalized treatment approach. Synergistic activity was demonstrated when fadraciclib was combined with the BCL-2 inhibitor venetoclax, or the conventional chemotherapy agents, cytarabine, or azacitidine, with the combination of fadraciclib and azacitidine having the most favorable therapeutic window. In summary, these results highlight the potential of fadraciclib as a novel therapeutic approach for AML.

Chronic myeloid leukaemia (CML) arises after transformation of a haemopoietic stem cell (HSC) by the protein-tyrosine kinase BCR-ABL. Direct inhibition of BCR-ABL kinase has revolutionized disease management, but fails to eradicate leukaemic stem cells (LSCs), which maintain CML. LSCs are independent of BCR-ABL for survival, providing a rationale for identifying and targeting kinase-independent pathways. Here we show--using proteomics, transcriptomics and network analyses--that in human LSCs, aberrantly expressed proteins, in both imatinib-responder and non-responder patients, are modulated in concert with p53 (also known as TP53) and c-MYC regulation. Perturbation of both p53 and c-MYC, and not BCR-ABL itself, leads to synergistic cell kill, differentiation, and near elimination of transplantable human LSCs in mice, while sparing normal HSCs. This unbiased systems approach targeting connected nodes exemplifies a novel precision medicine strategy providing evidence that LSCs can be eradicated., Competing Interests: Competing Interest Declaration–The work presented in Fig. 6 was in part supported by funding from Constellation Pharmaceuticals and Roche.

Targeting the Hedgehog (Hh) pathway represents a potential leukaemia stem cell (LSC)-directed therapy which may compliment tyrosine kinase inhibitors (TKIs) to eradicate LSC in chronic phase (CP) chronic myeloid leukaemia (CML). We set out to elucidate the role of Hh signaling in CP-CML and determine if inhibition of Hh signaling, through inhibition of smoothened (SMO), was an effective strategy to target CP-CML LSC. Assessment of Hh pathway gene and protein expression demonstrated that the Hh pathway is activated in CD34(+) CP-CML stem/progenitor cells. LDE225 (Sonidegib), a small molecule, clinically investigated SMO inhibitor, used alone and in combination with nilotinib, inhibited the Hh pathway in CD34(+) CP-CML cells, reducing the number and self-renewal capacity of CML LSC in vitro. The combination had no effect on normal haemopoietic stem cells. When combined, LDE225 + nilotinib reduced CD34(+) CP-CML cell engraftment in NSG mice and, upon administration to EGFP(+) /SCLtTA/TRE-BCR-ABL mice, the combination enhanced survival with reduced leukaemia development in secondary transplant recipients. In conclusion, the Hh pathway is deregulated in CML stem and progenitor cells. We identify Hh pathway inhibition, in combination with nilotinib, as a potentially effective therapeutic strategy to improve responses in CP-CML by targeting both stem and progenitor cells.

Modelling the parameters of multistep carcinogenesis is key for a better understanding of cancer progression, biomarker identification and the design of individualized therapies. Using chronic myeloid leukemia (CML) as a paradigm for hierarchical disease evolution we show that combined population dynamic modelling and CML patient biopsy genomic analysis enables patient stratification at unprecedented resolution. Linking CD34(+) similarity as a disease progression marker to patient-derived gene expression entropy separated established CML progression stages and uncovered additional heterogeneity within disease stages. Importantly, our patient data informed model enables quantitative approximation of individual patients' disease history within chronic phase (CP) and significantly separates "early" from "late" CP. Our findings provide a novel rationale for personalized and genome-informed disease progression risk assessment that is independent and complementary to conventional measures of CML disease burden and prognosis.

The effect on survival of including HLA-DPB1 in a 12-allele matching strategy was retrospectively evaluated in 130 patients with acute leukaemia and myelodysplasia undergoing T-cell-depleted PBSC transplantation using unrelated donors. Patients received alemtuzumab in vivo T-cell depletion as part of a myeloablative (MA; n=61) or reduced-intensity conditioning regimen (n=69). No difference in OS was seen with single-locus mismatching (mm) when 10 conventional alleles (HLA-A, B, C, DRB1 and DQB1) were considered. However, the addition of HLA-DPB1 matching data proved highly discriminatory. Mismatches were identified in 87% of patients previously considered fully matched (1DPmm=49pts: 2DPmm=28pts), and in the 9/10 group 22 patients were reclassified as double and 16 as triple mismatches. In 10/10 transplants, there was a distinct trend to poorer OS with double DPB1 mm. If all 12 loci were considered, 98% of single mm were at HLA-DPB1. Furthermore, cumulative mm at two or more loci was associated with significantly poorer 3-year OS (34% vs 48%, P=0.013: hazard ratio 1.8 (95% confidence interval 1.14-3.06; P=0.017), although his detrimental effect was only apparent using MA conditioning, in which reduced OS was associated with increased chronic GVHD (61% vs 16%, P=0.018) and nonrelapse mortality (30% vs 9%, P=0.039).

A blinded prospective study was performed to determine whether screening of whole blood using a real-time, panfungal polymerase chain reaction (PCR) technique could predict the development of invasive fungal infection (IFI) in immunocompromised haemato-oncology patients. In all, 78 patients (125 treatment episodes) were screened twice weekly by real-time panfungal PCR using LightCyclertrade mark technology. IFI was documented in 19 treatment episodes (five proven, three probable and 11 possible), and in 12, PCR was sequentially positive. PCR positivity occurred in: 4/5 proven; 2/3 probable; 6/11 possible; and 29/106 with no IFI. In 8/12 with IFI and sequentially positive PCR results, PCR positivity occurred before (median 19.5 days) and in 4/12 (median 10.5 days) after the initiation of empirical antifungal therapy. Based on sequential positive results for proven/probable IFI sensitivity, specificity, positive predictive value and negative predictive value were 75, 70, 15 and 98%, respectively. Real-time panfungal PCR is a sensitive tool for the early diagnosis of IFI in immunocompromised haemato-oncology patients. It may be most useful as a screening method in high-risk patients, either to direct early pre-emptive antifungal therapy or to determine when empirical antifungal therapy can be withheld in patients with antibiotic--resistant neutropenic fever. However, these strategies require further assessment in comparative clinical trials.