The gain of a second copy of the Philadelphia chromosome is one of the main secondary chromosomal changes related to the clonal evolution of cells with t(9;22) in chronic myelogenous leukemia. This gain causes the acquisition of another copy of the BCR/ABL1 fusion gene. Isochromosomes of the der(22) chromosome or double minute chromosomes are well known to lead an increased copy number of BCR/ABL1 gene. There is no antecedent of Philadelphia chromosome duplication as a ring chromosome. A recent published report contains evidence that strongly suggests that the Philadelphia chromosome was duplicated as a ring chromosome, observation that was overlooked by the authors. The instability inherent to the ring chromosome increases the risk of emergence of clones containing more and more BCR/ABL1 gene copies, which would produce increased fitness for clonal selection, resulting in worsening of the patient's prognosis.

Cyclic AMP-response element-binding protein 3-like 1 (CREB3L1) is a gene involved in the unfolded protein response (UPR). Recently, we demonstrated that CREB3L1 is specifically overexpressed in the platelets of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). In this study, we aimed to show the clinical and biological relevance of CREB3L1 in these hematological diseases. Overexpression of CREB3L1 was specific to platelets in MPNs and associated with a higher risk of thrombosis and fibrotic transformation in essential thrombocythemia (ET) and polycythemia vera (PV) cases, respectively. Furthermore, we found that UPR genes were downregulated in platelets of patients with ET and PV, which were more pronounced in patients harboring the JAK2 V617F mutation. However, CREB3L1 overexpression does not alter UPR gene expression or cell proliferation in UT-7/TPO/CALRm cells exogenously expressing mutated calreticulin and HEL cells harboring endogenous JAK2 V617F. Furthermore, CREB3L1 overexpression did not modulate sensitivity to endoplasmic reticulum stress in these cell lines. Taken together, our data show 1) a potential role of CREB3L1 expression in platelets as a new marker of high-risk MPNs and 2) an association between CREB3L1 overexpression and UPR gene downregulation in these patients' platelets, with CREB3L1 not altering UPR in our in vitro models and possibly further in vivo mechanisms being involved.

The emergence of tyrosine kinase inhibitors as part of a front-line treatment has greatly improved the clinical outcome of the patients with Ph+ acute lymphoblastic leukemia (ALL). However, a portion of them still become refractory to the therapy mainly through acquiring mutations in the BCR-ABL1 gene, necessitating a novel strategy to treat tyrosine kinase inhibitor (TKI)-resistant Ph+ ALL cases. In this report, we show evidence that RUNX1 transcription factor stringently controls the expression of BCR-ABL1, which can strategically be targeted by our novel RUNX inhibitor, Chb-M'. Through a series of in vitro experiments, we identified that RUNX1 binds to the promoter of BCR and directly transactivates BCR-ABL1 expression in Ph+ ALL cell lines. These cells showed significantly reduced expression of BCR-ABL1 with suppressed proliferation upon RUNX1 knockdown. Moreover, treatment with Chb-M' consistently downregulated the expression of BCR-ABL1 in these cells and this drug was highly effective even in an imatinib-resistant Ph+ ALL cell line. In good agreement with these findings, forced expression of BCR-ABL1 in these cells conferred relative resistance to Chb-M'. In addition, in vivo experiments with the Ph+ ALL patient-derived xenograft cells showed similar results. In summary, targeting RUNX1 therapeutically in Ph+ ALL cells may lead to overcoming TKI resistance through the transcriptional regulation of BCR-ABL1. Chb-M' could be a novel drug for patients with TKI-resistant refractory Ph+ ALL.

A major drawback of tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) is that primitive CML cells are able to survive TKI-mediated BCR-ABL inhibition, leading to disease persistence in patients. Investigation of strategies aiming to inhibit alternative survival pathways in CML is therefore critical. We have previously shown that a nonspecific pharmacological inhibition of autophagy potentiates TKI-induced death in Philadelphia chromosome-positive cells. Here we provide further understanding of how specific and pharmacological autophagy inhibition affects nonmitochondrial and mitochondrial energy metabolism and reactive oxygen species (ROS)-mediated differentiation of CML cells and highlight ATG7 (a critical component of the LC3 conjugation system) as a potential specific therapeutic target. By combining extra- and intracellular steady state metabolite measurements by liquid chromatography-mass spectrometry with metabolic flux assays using labeled glucose and functional assays, we demonstrate that knockdown of ATG7 results in decreased glycolysis and increased flux of labeled carbons through the mitochondrial tricarboxylic acid cycle. This leads to increased oxidative phosphorylation and mitochondrial ROS accumulation. Furthermore, following ROS accumulation, CML cells, including primary CML CD34(+) progenitor cells, differentiate toward the erythroid lineage. Finally, ATG7 knockdown sensitizes CML progenitor cells to TKI-induced death, without affecting survival of normal cells, suggesting that specific inhibitors of ATG7 in combination with TKI would provide a novel therapeutic approach for CML patients exhibiting persistent disease.

As one of the major treatment obstacles in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL), relapse of Ph+ALL may result from the persistence of leukemia-propagating cells (LPCs). Research using a xenograft mouse assay recently determined that LPCs were enriched in the CD34+CD38-CD58- fraction in human Ph+ALL. Additionally, a cohort study demonstrated that Ph+ALL patients with a LPCs phenotype at diagnosis exhibited a significantly higher cumulative incidence of relapse than those with the other cell phenotypes even with uniform front-line imatinib-based therapy pre- and post-allotransplant, thus highlighting the need for novel LPCs-based therapeutic strategies.

Epigenetic silencing of sFRP genes has been shown to lead to constitutive activation of the canonical Wnt-signaling pathway. The first description of deregulated Wnt-signaling activation in a hematological malignancy was reported in chronic myeloid leukemia (CML). To investigate whether epigenetic silencing of sFRP is responsible for the observed Wnt activation in CML, we studied the methylation and mutational status of the sFRP1 promoter in 48 chronic phase CML patients. Of the 48 CML patients 41 were shown to be unmethylated, 6 patients hemi-methylated and 1 patient fully methylated at the sFRP1 promoter. Albeit observed infrequently in chronic phase CML, sFRP1 promoter methylation correlated with primary cytogenetic resistance to imatinib mesylate. sFRP1 promoter methylation may indicate a genetically more unstable form of disease resistant to therapy and provide a key biological difference in therapy resistant patients, in addition to a possible mechanism for the observed activation of canonical Wnt signaling in CML.

Discrimination of Philadelphia-negative myeloproliferative neoplasms (Ph-MPNs) from reactive hypercytosis and myelofibrosis requires a constellation of testing including driver mutation analysis and bone marrow biopsies. We searched for a biomarker that can more easily distinguish Ph-MPNs from reactive hypercytosis and myelofibrosis by using RNA-seq analysis utilizing platelet-rich plasma (PRP)-derived RNAs from patients with essential thrombocythemia (ET) and reactive thrombocytosis, and CREB3L1 was found to have an extremely high impact in discriminating the two disorders. To validate and further explore the result, expression levels of CREB3L1 in PRP were quantified by reverse-transcription quantitative PCR and compared among patients with ET, other Ph-MPNs, chronic myeloid leukemia (CML), and reactive hypercytosis and myelofibrosis. A CREB3L1 expression cutoff value determined based on PRP of 18 healthy volunteers accurately discriminated 150 driver mutation-positive Ph-MPNs from other entities (71 reactive hypercytosis and myelofibrosis, 6 CML, and 18 healthy volunteers) and showed both sensitivity and specificity of 1.0000. Importantly, CREB3L1 expression levels were significantly higher in ET compared with reactive thrombocytosis (P < .0001), and polycythemia vera compared with reactive erythrocytosis (P < .0001). Pathology-affirmed triple-negative ET (TN-ET) patients were divided into a high- and low-CREB3L1-expression group, and some patients in the low-expression group achieved a spontaneous remission during the clinical course. In conclusion, CREB3L1 analysis has the potential to single-handedly discriminate driver mutation-positive Ph-MPNs from reactive hypercytosis and myelofibrosis, and also may identify a subgroup within TN-ET showing distinct clinical features including spontaneous remission.

Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukaemia (ALL), a high-risk subtype characterised by genomic alterations that activate cytokine receptor and kinase signalling, is associated with inferior outcomes in most childhood ALL clinical trials. Half of the patients with Ph-like ALL have kinase rearrangements or fusions. We examined the frequency and spectrum of these fusions using a retrospective cohort of 212 newly diagnosed patients with childhood B-cell ALL. Samples without known chromosomal alterations were subject to multiplex reverse transcription polymerase chain reaction to identify known Ph-like kinase fusions. Immunoglobulin heavy chain locus (IGH) capture and kinase capture were applied to samples without known kinase fusions. We detected known kinase fusions in five of 212 patients, comprising EBF1-PDGFRB, ETV6-ABL1, ZC3HAV1-ABL2, EPOR-IGH, and CNTRL-ABL1. Two patients with P2RY8-CRLF2 were identified. Patients with non-Ph kinase fusions had inferior 5-year event-free survival and overall survival compared with patients with other common genetic alterations. The prevalence of non-Ph kinase fusions in our Taiwanese cohort was lower than that reported in Caucasian populations. Future clinical trials with tyrosine kinase inhibitors may be indicated in Taiwan because of the inferior outcomes for B-cell ALL with kinase fusions.

Essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) are clonal hematological diseases classified as Philadelphia chromosome-negative myeloproliferative neoplasms (MPN). MPN pathogenesis is associated with the presence of somatic driver mutations, bone marrow (BM) niche alterations, and tumor inflammatory status. The relevance of soluble mediators in the pathogenesis of MPN led us to analyze the levels of cytokines, chemokines, and growth factors related to inflammation, angiogenesis and hematopoiesis regulation in the BM niche of MPN patients.

Chronic myelogenous leukemia (CML) is an indolent malignant hematological disease that accounts for about 15% of all cases of leukemia. This disorder results from the formation of the Philadelphia chromosome that involves a reciprocal translocation that produces a lengthened chromosome 9 and shortened chromosome 22 - the Philadelphia chromosome. As a consequence of the translocation, the dysregulated BCR-Abl fusion oncoprotein is formed and it produces the abnormal proliferation of white blood cells. The treatment of CML with imatinib revolutionized the treatment of this disorder and led to the discovery and development of dozens of effective targeted protein kinase inhibitors. Imatinib (first generation), dasatinib, nilotinib, and bosutinib (second generation) have been FDA-approved for frontline therapy, and ponatinib (third generation) is approved for resistant disease with a T315I mutation. Each of these drugs is orally bioavailable. The BCR-Abl fusion protein lacks the physiological N-terminal myristoyl group that binds to a hydrophobic pocket in the large protein kinase lobe and inhibits enzyme activity. The absence of the myristoyl group leads to enhanced protein kinase catalytic activity. Asciminib was designed to bind to this binding pocket to reduce Abl kinase activity. Asciminib is orally effective and was FDA-approved as a third-line treatment for CML and a first-line treatment in patients with the T315I mutation. It blocks the activity of BCR-Abl by interacting with the myristate-binding site located 23 Å from the ATP-binding site and is the prototype of a type IV inhibitor. Asciminib is a so-called STAMP inhibitor that Specifically Targets the Abl Myristoyl Pocket.

To shed light onto the molecular basis of Philadelphia chromosome-positive acute lymphoblastic leukemia and to investigate the prognostic role of additional genomic lesions, we analyzed copy number aberrations using the Cytoscan HD Array in 116 newly diagnosed adult patients with Philadelphia chromosome-positive acute lymphoblastic leukemia enrolled in four different GIMEMA protocols, all based on a chemotherapy-free induction strategy. This analysis showed that patients with Philadelphia chromosome-positive acute lymphoblastic leukemia carry an average of 7.8 lesions/case, with deletions outnumbering gains (88% versus 12%). The most common deletions were those targeting IKZF1, PAX5 and CDKN2A/B, which were detected in 84%, 36% and 32% of cases, respectively. Patients carrying simultaneous deletions of IKZF1 plus CDKN2A/B and/or PAX5 had a significantly lower disease-free survival rate (24.9% versus 43.3%; P=0.026). The only IKZF1 isoform affecting prognosis was the dominant negative one (P=0.003). Analysis of copy number aberrations showed that 18% of patients harbored MEF2C deletions, which were of two types, differing in size: the longer deletions were associated with the achievement of a complete molecular remission (P=0.05) and had a favorable impact on disease-free survival (64.3% versus 32.1% at 36 months; P=0.031). These findings retained statistical significance also in multivariate analysis (P=0.057). KRAS deletions, detected in 6% of cases, were associated with the achievement of a complete molecular remission (P=0.009). These results indicate that in adults with Philadelphia chromosome-positive acute lymphoblastic leukemia a detailed evaluation of additional deletions - including CDKN2A/B, PAX5, IKZF1, MEF2C and KRAS - has prognostic implications and should be incorporated in the design of more personalized treatment strategies.

Since BCR-ABL plays an essential role in the growth factor-independent proliferation of Philadelphia chromosome (Ph)+ leukemia cells, imatinib treatment of Ph+ leukemia cells inactivates signaling pathways of BCR-ABL, and subsequent addition of growth factors (GFs) could restore the signaling pathways without reactivating BCR-ABL. Here we demonstrated that non-lymphoid Ph+ leukemia cell lines responded to diverse GFs depending on their immunophenotype and gene expression of transcription factors and GF receptors, while lymphoid Ph+ leukemia cell lines restrictively responded to flit3 ligand and interleukin-7, suggesting that GF sensitivity of imatinib-treated Ph+ leukemia cells could be powerful for specifying their distinctive lineage.

Since myeloproliferative neoplasms (MPN) pose a significant risk for vascular and thrombotic complications, cytoreductive therapies, such as hydroxyurea (HU), interferon (IFN) inhibitors, and Janus kinase (JAK) inhibitors are recommended for patients at high risk. However, these agents also place patients at increased risk for drug-related cutaneous adverse events. Herein, we review the literature on skin toxicity related to the use of drugs for the treatment of MPN. Overall, the cytoreductive agents used for MPN are generally well tolerated and considered to be safe, except IFN, for which dropout rates as high as 25% have been reported. While IFN is known to give rise to flu syndrome, it rarely leads to hematological alterations. The most common hematological side effects of HU are mild and include granulocytopenia, anemia, and thrombocytopenia. The JAK inhibitor ruxolitinib has been associated with cytopenia and a higher incidence of viral infections, as well as increased risk for basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Based on the present analysis, it can be concluded that cutaneous toxicity is not a negligible complication of commonly used treatments for MPN. While further research is needed, patients on these agents, and especially those with a history of cutaneous malignancies, should undergo thorough skin examination before and during therapy. In addition, detailed history is critical since many patients who develop non-melanoma skin cancer have multiple preexisting risk factors for cutaneous carcinogenesis.

The Philadelphia chromosome (Ph) resulting from the t(9;22) translocation generates the oncogenic BCR::ABL1 fusion protein that is most commonly associated with chronic myeloid leukemia (CML) and Ph-positive (Ph+) acute lymphoblastic leukemia (ALL). There are also rare instances of patients (≤1%) with newly diagnosed acute myeloid leukemia (AML) that harbor this translocation (Paietta et al., Leukemia 12: 1881 [1998]; Keung et al., Leuk Res 28: 579 [2004]; Soupir et al., Am J Clin Pathol 127: 642 [2007]). AML with BCR::ABL has only recently been provisionally classified by the World Health Organization as a diagnostically distinct subtype of AML. Discernment from the extremely close differential diagnosis of myeloid blast crisis CML is challenging, largely relying on medical history rather than clinical characteristics (Arber et al., Blood 127: 2391 [2016]). To gain insight into the genomic features underlying the evolution of AML with BCR::ABL, we identified a patient presenting with a high-risk myelodysplastic syndrome that acquired a BCR::ABL alteration after a peripheral blood stem cell transplant. Serial samples were collected and analyzed using whole-exome sequencing, RNA-seq, and ex vivo functional drug screens. Persistent subclones were identified, both at diagnosis and at relapse, including an SF3B1p.Lys700Glu mutation that later cooccurred with an NRASp.Gly12Cys mutation. Functional ex vivo drug screening performed on primary patient cells suggested that combination therapies of ABL1 with RAS or PI3K pathway inhibitors could have augmented the patient's response throughout the course of disease. Together, our findings argue for the importance of genomic profiling and the potential value of ABL1 inhibitor-inclusive combination treatment strategies in patients with this rare disease.

Submicroscopic segmental imbalances detected by array-comparative genomic hybridization (array-CGH) were discovered to be common in chronic myeloid leukemia (CML) patients with t(9;22) as the sole chromosomal anomaly. To confirm the findings of the previous study and expand the investigation, additional CML patients with t(9;22) as the sole chromosomal anomaly were recruited and copy number variants (CNVs) were searched for.

Targeted therapy has revolutionized the management of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL); however, relapse still occurs because of the presence of quiescent stem cells, termed leukemia propagating cells (LPCs). This study aimed to assess the phenotypic diversity of LPCs in adult patients with Ph+ B-Acute ALL (B-ALL) and to assess its prognostic impact.

Dysregulated hematopoietic niches remodeled by leukemia cells lead to imbalances in immunological mediators that support leukemogenesis and drug resistance. Targeting immune niches may ameliorate disease progression and tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive B-ALL (Ph+ B-ALL). Here, we show that T helper type 17 (Th17) cells and IL-17A expression are distinctively elevated in Ph+ B-ALL patients. IL-17A promotes the progression of Ph+ B-ALL. Mechanistically, IL-17A activates BCR-ABL, IL6/JAK/STAT3, and NF-kB signalling pathways in Ph+ B-ALL cells, resulting in robust cell proliferation and survival. In addition, IL-17A-activated Ph+ B-ALL cells secrete the chemokine CXCL16, which in turn promotes Th17 differentiation, attracts Th17 cells and forms a positive feedback loop supporting leukemia progression. These data demonstrate an involvement of Th17 cells in Ph+ B-ALL progression and suggest potential therapeutic options for Ph+ B-ALL with Th17-enriched niches.

ABL tyrosine kinase inhibitor (TKI) therapy has improved the survival of patients with Philadelphia (Ph) chromosome-positive leukemia. However, ABL TKIs cannot eradicate leukemia stem cells. Therefore, new therapeutic approaches for Ph-positive leukemia are needed. Aberrant activation of phosphoinositide 3-kinase (PI3K) signaling is important for the initiation and maintenance of human cancers. Copanlisib (BAY80-6946) is a potent inhibitor of PI3Kα and PI3K-δ. Here we investigated the efficacy of combination therapy of copanlisib with an ABL TKI (imatinib, nilotinib, or ponatinib) using BCR-ABL-positive cells. Although the effects of the ABL TKI treatment were reduced in the presence of the feeder cell line, HS-5, copanlisib inhibited cell growth. Upon combining ABL TKI and copanlisib, cell growth was reduced. Ponatinib and copanlisib combined therapy reduced tumor volume and increased survival in mouse allograft models, respectively. These results indicate that the PI3Kα and -δ inhibitors overcame the chemoprotective effects of the feeder cells and enhanced ABL TKI cytotoxicity. Thus, co-treatment with ABL TKI and copanlisib may be a powerful strategy against ABL TKI-resistant cells, including those harboring the related T315I mutation.

Chronic myeloid leukemia (CML) is genetically characterized by the presence of the reciprocal translocation t(9;22) with the formation of Philadelphia (Ph) chromosome. Sometimes, the Ph translocation is generated by variant rearrangements. The prognostic impact of the variant translocations is still controversial. Among the 180 patients with Ph-positive CML who were treated in Hacettepe University Faculty of Medicine Division of Hematology, variant translocations were detected, and retrospectively clinical and prognostic features were described. Also we performed a comprehensive literature review on the prognosis of such variant cases before and after tyrosine kinase inhibitor era. Five patients (2.7%) had variant Ph chromosomes, involved in the rearrangements were chromosomes 2 (2 cases), 11, 14 and 15. Patients were treated with imatinib or dasatinib. All patients reached a stable major molecular response suggesting a prognosis not worse than standard translocation individuals. Our present data were compatible with the data of previous studies indicating no difference in the prognosis between standard and variant translocations in tyrosine kinase inhibitors era of CML.

A clonal cytogenetic abnormality was observed in Philadelphia chromosome-negative bone marrow cells of 6/27 chronic myeloid leukemia patients (+8 in 4, -7 in 1, and 20q- in 1) with dasatinib-induced remissions. The X-linked human androgen receptor gene assay demonstrated clonality in one additional patient. Single nucleotide polymorphism array analysis revealed somatic uniparental disomy involving chromosome 17(p12-pter) in another patient. The TP53 gene had a 5' splice site deletion of exon 6 that caused alternative splicing, frame shifting and introduction of a premature stop codon. After three years, no patient developed myelodysplastic syndrome or acute myeloid leukemia.